Acta path. microbiol. scand. Sect. B. 86: 303-307, 1978.

EFFECT OF HUMAN LEUKOCYTE INTERFERON ON THE PERMEABILITY OF THE CYTOPLASMA MEMBRANE

OF CULTURED CELLS

MIKLOS DEGRk

Kapt. W. Wilhelmsen og Frues Bakteriologiske Institutt, University of Oslo. Rikshospitalet. Oslo. Norway

DegrC, M. Effect of human leukocyte interferon on the permeability of the cytoplasma membrane of cultured cells. Acta path. microbiol. scand. Sect. B. 86: 303-307. 1978.

The effect of human leukocyte interferon on the cytoplasrna membrane of cultured homologous cells has been investigated. U-amnion cells were labelled with (I-14C) alpha-aminoisobutyric acid (AIB), a low-molecular-weight non-metabolizable amino acid. Following uptake, the AIB is released spontaneo- usly from the cells. Treatment of cells with interferon reduced the AIB release. This effect was dosedependent, was neutralized by anti-interferon serum and was strongly reduced by trypsin treatment.

Key words: interferon; cytoplasma membrane.

M. Degre, Kapt. W. Wilhelmsen og Frues Bakteriologiske Institutt, Rikshospitalet. Oslo I , Norway.

Received 5.v.78 Accepted 6.vi.78 An effect on the cytoplasrna membrane is

probably a crucially important part of the action of interferon on homologous cells. It has been reported by several groups of investigators that in some viruscell systems interferon inhibits a late stage in virus production, assembly, maturation or release of virus particles ( I , 2, 1 1 . 15, 16). While the intracellular markers of e.g. murine leukaemia virus are produced normally, complete virus partic- les are released in reduced numbers from interferon- treated cells ( I I ). It has been suggested that this might be a result of an interferon-induced alteration of cell membrane physiology.

To investigate the interferon effect on the stability of the cell membrane, we employed 3H labelled uridine as a low-molecular-weight cytoplasmic marker (10). Treatment of human U-amnion cells with low concentrations of human leukocyte interferon reduced the spontaneous release of 3H- uridine from the cells, thus supporting the theory that interferon treatment stabilizes the cell memb- rane. Since the publication of that report, an

improvement of this method has been described by 7'helesrum & Mollby (171, using (I-'%) alpha- aminoisobutyric acid (AIB). This compound is a low-molecular-weight (mo1.m. 1 03) non-metaboli- zable amino acid, and is therefore a good cytoplas- mic marker. It has been shown that the sensitivity of the assay for membrane stability is high.

In the present study we have investigated the effect of human interferon on the spontaneous release ,of AIB from cultured homologous cells.

MATERIALS AND METHODS

Cells The U line of human amnion cells was originally

received from Dr K . Cantell. Helsinki: The cells were grown in Eagle's minimal essential medium (MEM, GIBCO) with the addition of 5 per cent inactivated calf serum, NakiC03 and antibiotics. The cells were main- tained in the same medium with 2 per cent calf serum.

Human embryonic lung cells (HEL) were prepared in this laboratory. They were grown in 50 per cent Eagle's MEM and 50 per cent medium 199 (GIBCO), with the addition of 10 per cent calf serum, NaHCO3 and

303

antibiotics. The serum concentration was reduced to 5 and later to 2 per cent during maintenance. The HEL cells were in their 5th to 15th passage during the present study.

virus Vesicular stomatitis virus (VSV). Indiana strain, was

grown in L-FI mouse fibroblast cells. Samples were stored at -7OO C and a fresh ampoule was used for each experiment. Infectivity titres were assayed by the infectivity endpoint micromethod in L-FI and U cells.

Interferon Human leukocyte interferon preparations were kindly

provided by Dr. K . Cantell, Helsinki. Both crude interferon and partially purified preparations (4) were employed. The results obtained with the different preparations were identical and therefore are not presented separately. Mock interferon preparations were also obtained from Dr. K . Cantell. Mock interferons were prepared in the same way as the leukocyte interferons. except for omission of the Sendai virus induction of the leukocytes.

Anti-interferon Serum Rabbit anti-human globulin against leukocyte interfe-

ron was received from the Antiviral Substances Pro- gram, National Institute of Allergy and Infectious Diseases, Bethesda, Md.

Assay of the Biological Effect of Integeron Antiviral activiw of interferon preparations was tested

by the infectivity inhibition micromethod, employing VSV and HEL cells. Details of the test are described elsewhere (8). Titres are expressed in international standard units related to the 69 / I9 standard.

Integeron eflect on cell growth was tested as described in detail elsewhere (9). Briefly, I ml of medium containing interferon dilutions was added to freshly seeded U cells, IOS cells per tube. At least five tubes were used for each dilution. Control tubes were seeded with medium without interferon. The cells were incubated stationary in 5 per cent COz atmosphere at 37O C. After incubation for 3 days the monolayers were trypsinized and the cells were counted in a haemoqmmeter. Viability was more than 95 per cent. as determined by the trypan blue staining method.

Interferon Eflect on Relase of AIB (I--14C) alpha-aminoisobutyric acid (AIB) was pur-

chased from The Radio-Chemical Centre, Amersham. Stock solutions made in Hanks' BS were kept at -2OO C. Tubes were seeded with 105 U cells each in MEM. Three-day-old monolayers were incubated with AIB diluted in MEM to contain 1 pCi per ml. After incubation for 1 hour, the cells were washed three times with fresh medium to remove any radioactivity not incorporated into the cells. Interferon diluted in MEM was then added, using 5-8 tubes for each dilution. MEM alone was added to the control tubes. After incubation at 37OC for various times, 0.1 ml of the supernatant was transferred

to a scintillation vial containing 10 ml Aerosol. Samples were counted in a Packard liquid scintillator for 10 minutes. The activity in the control culture supernatants was considered as 100 per cent, and the interferon treated cultures were related to the controls.

In some experiments, the remaining medium was decanted and the cells were suspended in 0.9 ml fresh MEM and 0.1 ml of the non-ionic detergent Triton-X 100 at a final concentration of 0.25 per cent (v/v). The tubes were incubated for 15 minutes and agitated in a Vortex mixer. This treatment causes complete lysis of the cytoplasma membrane of the cells, thus releasing the incorporated radioactivity into the medium (18). After lysis, 0.1 ml was transferred to a scintillation vial containing Aerosol and the radioactivity was determined as described above. The total radioactivity taken up by the cells was equal to the sum of released and cell-bound radioactivity.

RESULTS

Spontaneous Release of AIB from Normal U Cells Three-day-old monolayers of U cells were

labelled with AIB as described in Materials and Methods. The amounts of extra-cellular and cell- bound radioactivity were determined after various periods of incubation. Results of a representative experiment are shown in Fig. I . After uptake, some of the radioactivity was released spontaneously from the labelled cells into the medium. The total radioactivity, i.e. the sum of extracellular and cellbound radioactivity, was constant during the time of the experiment. The kinetics of spontaneous release were similar in several repeated experiments.

Interferon E/fecr on Spontaneous Release of AIB from Normal U Cells

200 units interferon per ml diluted in MEM were added to the cells immediately after removal of the radioactive AIBcontaining medium. Control tubes were supplemented with MEM only. After incuba-

, / , , 1

I aI 4 1 I. 1 8 "

Fig. I . Spontaneous release of AIB from U-amnion cells. Vertical bars indicate standard deviation (SD).

304

tion for IS, 30, 45 and 90 minutes, 0.1 ml was removed from the supernatant and the radioactivity was determined (Fig. 2). The supernatant of the interferon treated cultures contained less radioacti- vity after incubation for 45 and 90 minutes than the supernatant of the control cultures.

TABLE I . Eflect of Yarious Concentrations of Interferon on the Spontaneous Release of AIB from U Cells

Interferon Units Counts Percentage per ml per minute f SD of control

0 801 f 4 1 5 832 f 55 I04

10 164 f 61 95 50 164 f 82 95

200 739*10 92 1000 614 f 54 84

AIB release was measured after incubation for 90 minutes.

ron. Higher concentrations of interferon (200 units per ml or more) reduced the extracellular radioacti- vity.

L I, I. , I. 1 1 "

Specijcity of the Suppressing Eflect of Interferon The specificity of the interferon effect was tested

by exposing the interferon to treatment with physico-chemical factors known to have well- defined effects on the biological activities of interferon. Trypsin treatment is known to eliminate the antiviral effect, while heating at 5 6 O C for 30 minutes has no major influence. In addition, specific antiserum is known to neuralize both the antiviral

Fig. 2 . Effect of human leukocyte interferon on the spontaneous release of AIB from U amnion cells. V e ~ c a l bars indicate 'Standard deviation (SD).

The interferon effect on the AIB release was dose dependent (Table I). A slight increase (105-1 15 per cent of the controls) was seen regularly after treatment with very low concentrations of interfe-

TABLE 2. Specijiny of Interferon Eflecf on the Cell Membrane Permeability

Number of cells x 10.000fSD *

Release of AIB 96 of total b

titre ( % of controls) radioactivity

Antiviral

Experiment 1

Control cells Interferon Interferon treated at 56O C for 30 min. Interferon treated with trypsin

Experiment I1 control cells Interferon Mock interferon Interferon treated with antiinterferon globulin

I584 I000

I25

I60 < 10 < 10

41 f 8.2 22 f 4.5 (56) 21 f 3 . 2 (51)

37 f 9.9 (90)

21 f 3.3 1 I f 0.9 (52) 18 f4 .5 (85) 20 f 3.9 (95)

18 58 53

68

38 29 39 39

a Number of cells after incubation for 3 days with the test substance. AIB release was measured after incubation for 90 minutes. Interferon was incubated with anti-interferon globulin overnight.

305

and non-antiviral activities of interferon. Following treatment of interferon, samples were tested for effect on AIB release. Antiviral activity and effect on cell growth were determined in parallel experi- ments (Table 2). 1500 units interferon per ml reduced the AIB release by 24 per cent and the cell growth by 44 per cent. Heating had little if any effect on the activities, while trypsin treatment strongly reduced all three activites. Anti-interferon globulin had no effect by itself and had no influence on the effect of the low interferon concentrations (1-5 units) on the AIB release. When provoked by 200 units interferon, all three activities were neutralized by treatment with anti-interferon globu- lin. Mock interferon showed no significant activity when tested by the same three parameters.

DISCUSSION

AIB was chosen as a cytoplasmic marker mainly because it is not metabolized in the cell (7). Therefore its release from the cells must be primarily dependent on the state of the cytoplasma membrane. The release of AIB is also dependent on the solvent in which the cells are incubated. Eagle's MEM was chosen for the present experiments because the spontaneous release into this medium is more pronounced than into other solutions (1 7). Therefore a possible reduction of the release by stabilization of cell membrane could be more easily demonstrated. The extent of spontaneous release of AIB from U amnion cells was comparable to that found by others (17) from human embryonal fibroblast cells.

The present results show that human leukocyte interferon inhibits the spontaneous release of a non- metabolizable cytoplasmic marker from the homo- logous U amnion cells. This effects was dose- dependent and correlated to the antiviral and cell growth inhibitory activities of the interferon prepa- ration. Furthermore, the effect seems to be specific and is eliminated by trypsin treatment and anti- interferon treatment of interferon preparation. This finding confums the earlier results obtained with 3H-uridine as cytoplasmic marker ( 1 0). The findings indicate that the reduction of release is not specific for the one marker used in the earlier experiments.

There is considerable experimental evidence suggesting that interferon treatment caw modifi- cations of the cell surface. Murine leukaemia cells produced a reduced number of complete extracellu- lar virus particles, while several intracellular markers were not influenced (1 I). A marked accumulation of virus particles at the surface indicated that the blockmg effect is effective at a late stage of virus production, probably connected with

the release process (5). Signs of altered surface chareristia were also observed in various ceh without the presence of virus infection. Such alterations were registered by a number of different parameters, such as expression of histocompatibility antigens (1 41, altered binding of radioactive conca- navalin A ( 1 21, cholera toxin and tyreoideastimula- ting hormone (TSH) (1 3) to the cell surface, effect on the transport of thymidine (31, effect on the spontaneous release of uridine ( 101, alteration of the density of membrane and presence of intramem- brane granules (6). The present findings are com- patible with these observations and confirm that interferons alter the cytoplasma membrane.

The quantitative aspects of the present study differ somewhat from our earlier findings, since larger concentrations of interferon were necessary to reduce the release of AIB than that of 3H-uridine (10). Since the AIB release can be considered as a marker of cytoplasma membrane permeablility, the Merence might suggest that intracellular events involving uridine metabolism might be influenced by the interferon treatment.

Very low concentrations of interferon ( 1-5 units per ml) in several experiments stimulated slightly AIB release from the cells. However, this effect was not neutralized by the specific antiserum, and therefore should probably be considered as non- specific.

AIB was originally introduced in order to study the effect of cytolytic substance on the membrane permeability (1 7). The present results indicate that this substance may also be applied to the study of opposite effects, i.e. stabilization of cell membrane.

The excellent technical assistance of Zdenka Krajs is gratefully acknowledged.

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