VIROLOGY

99,

Interferon

158-166 (1979)

Production by Human Lymphoblastoid Cells is Stimulated by Inducers of Friend Cell Differentiation GUENTHER BiocLmical

Laboratory,

R. ADOLF

AND PETER

Ernst Boehringer A-1121 Vienna, Accepted

Znstitut Austria

August

fuer

SWETLY’

Arzneimittelforschung,

I, 1979

A variety of structurally unrelated substances stimulated production of human lymphoblaatoid interferon in Namalwa cells. Active substances include short-chain fatty acids, dimethyl sulfoxide, and hexamethylene bisacetamide. These substances are established inducers of erythropoietic differentiation in mouse erythroleukemic spleen cells (Friend cells). When Namalwa cells were cultured for 24 hr or more in the presence of l-2.5 n&f n-butyric acid and then induced with Sendai virus, a 30-fold increase in interferon yields over untreated Namalwa cells was observed. Propionic acid and n-valeric acid at a concentration of 5 m&f caused a similar increase in interferon production and eaproic acid at 5 m&f stimulated it S-fold. Substituted or unsaturated fatty acids were inert. Dimethyl sulfoxide increased interferon production at 280 rn% and hexamethylene bisaeetamide at 10 mAf. All substances which enhanced interferon production blocked thymidine incorporation into Namalwa cell DNA at concentrations equal to those effective in interferon stimulation. INTRODUCTION

Sodium n-butyrate in low concentrations causes a variety of reversible changes in mammalian cells in culture (reviewed by Prasad and Sinha, 1976). Besides effects on cell proliferation and morphology in a number of cell types, n-butyrate is able to induce the synthesis of specialized proteins, including peptide hormones (Ghosh and Cox, 1976; Ghosh and Cox, 1977) in HeLa cells, enzymes of neurotransmitter metabolism in neuroblastoma cells (Prasad and Sinha, 1976), and Padrenergic receptors in HeLa cells (Tallman et al., 1977). Perhaps most interestingly, n-butyrate also was shown to be a potent inducer of cell differentiation in mouse erythroleukemia cells (Friend cells), stimulating differentiation of these cells into hemoglobin-containing, nondividing cells with many characteristics of normoblasts (Leder and Leder, 1975). Besides butyrate, a variety of seemingly unrelated compounds including dimethyl sulfoxide (Friend et al., 1971, Ostertaget al., 1972) and hexamethylene bisacetamide (Reubenet al., 1976) were

shown to be potent inducers of Friend cell differentiation, although their activities vary to some extent with different Friend cell lines. We attempted to study the effects of a number of known inducers of specialized proteins on interferon production in Namalwa cells. Cells of the human lymphoblastoid line Namalwa, derived from a Burkitt lymphoma and containing integrated Epstein-Barr virus sequences, are often used as a source of human lymphoblastoid interferon (Strander et al., 1975; Bridgen et uZ., 1977; Have11 et al., 1978). We report here that n-butyrate and other inducers of hemoglobin synthesis in Friend cells block the proliferation of Namalwa cells and forcefully increase the ability of the cells to generate interferon upon stimulation with Sendai virus. This principle can be applied for the large scale production of lymphoblastoid interferon (Swetly et al., 1979). MATERIALS

Cells and viruses. Namalwa cells and Friend cells (F4N; Ostertag et al., 1972)

L To whom reprint requests should be addressed. 0042-6822/79/15015-9-09$02.00/O Copyright All rights

Q 1979 by Academic Press, Inc. of reproduction in any form reserved.

AND METHODS

158

ENHANCEMENT

OF INTERFERON TABLE

PRODUCTION

159

1

INTERFERON PRODUCTIONBY CELLSTREATEDWITHSHORT-CHAIN

FA~YACIDS~ Interferon titer Relative increase

Concentration m4 Acetic acid

Number of experiments

Range (unit&O6 cells)

Range

Mean

1 5

1 3

130 210-460

l-3

1 2

Propionic acid

1 5

3 2

130-2,500 2,400-18,600

2-5 28-38

4 33

n-Butyric

acid

1

7

1,500-15,500

13-37

29

n-Valerie acid

1 5

2 3

200-2,500 1,400-7,900

2-5 12-3’7

4 22

n-Caproic acid

5

2

180-700

7-9

8

a Rapidly growing cells were suspended in fresh growth medium at 5-7.105 cells/ml and the compounds indicated were added to the cultures as their respective sodium salts. After 48 hr, interferon production was induced by Sendai virus as described under Materials and Methods. The ratios of interferon titers of treated and untreated parallel cultures are given as proportional increase.

were kept in suspension culture in Eagle’s minimum essential medium (MEM) containing twofold quantities of amino acids and vitamins and 10% fetal calf serum (GIBCO). Cell densities were measured by hemocytometer counting and viability was determined by trypan blue exclusion. The proportion of hemoglobin-containing F4N cells was established 48 hr after addition of the stimulating substance to the culture medium by liquid benzidine staining (Orkin et al., 1975). Vero cells were grown in Medium 199 (Earle’s salts) containing 5% fetal calf serum. Sendai virus and vesicular stomatitis virus (strain Indiana) were gifts of Dr. G. Bodo. Interferon induction and assay. Cells grown with or without stimulating substances were collected by centrifugation, resuspended in MEM containing 2% fetal calf serum at cell densities from 5 to 15. lo6 cells/ml, and incubated with 21° hemagglutinating units of Sendai virus/ml for 2 hr at 37” under constant shaking. Cells were then pelleted (1000 g, 5 min), washed once with serum-free MEM, and suspended in the same medium at concentrations from 5 to 15 * lo5 cells/ml. After 20 hr at 37”, cell supernatants were harvested and held at pH 2

and 4” for 4 days to destroy residual inducer virus. Interferon titers were defined as the reciprocal of the cell supernatant dilution necessary to reduce by 50% the plaque count of vesicular stomatitis virus on Vero cells. All interferon titers were expressed in terms of the international reference standard leukocyte interferon 69/19. Measurement of DNA synthesis. After determination of cell densities, l-ml samples were incubated at 37” for 15 min with 3 $W ml of [3H]thymidine (47.5 Wmmol, The Radiochemical Centre, Amersham), chilled rapidly in an ice bath to terminate incorporation, the cells collected by centrifugation, and the radioactivity incorporated into DNA was determined by precipitation with 7% trichloroacetic acid, filtration, and liquid scintillation counting. RESULTS

Stimulation of Interferon Short-Chain Aliphatic

Production Acids

by

In an attempt to augment interferon production in Namalwa cells we tested the effect of pretreatment of cells with n-butyric

160

ADOLF

AND

acid or analogous saturated aliphatic monocarbonic acids. Namalwa cells were incubated for 48 hr at 5.105 cells/ml in culture medium supplemented with one short-chain fatty acid and subsequently induced for interferon production with a constant amount of Senclai virus. Fatty acids were used at concentrations between 1 and 5 mM; above 5 mlLl several compounds tested displayed cytotoxic effects. The results of such experiments on interferon production per unit number of cells are summarized in Table 1. Exposure of Namalwa cells to 1 n-d! n-butyric acid leads to a mean 29-fold increase in interferon production; in a series of seven independent experiments, the relative increase ranged from 13- to 37-fold. Higher concentrations of n-butyric acid caused no further enhancement of interferon production. At 1 mM all other fatty acids tested amplified interferon production only up to 4-fold.

Resolution of the enhancement effect in terms of carbon chain length of fatty acids is illustrated in Fig. 1. Maximal increase is reached at carbon chain length 4 (C4) at 1 mM with a marked fall on both sides for C3 and C5. At 5 n&l, C3 and C5 attain the same level as C4 at 1 n-d4 and C2 and C6 are notably less effective. In order to determine whether metabolic intermediates of short-chain fatty acids of the acids themselves mediated the enhancement effect we tested C4 acids with hydrophilic substitutions, a branched and an un-

SWETLY

4 Fatty

OF DIFFERENT

FOUR-CARBON

cha,n

5

6

lsngth

FIG 1. Dependence of interferon production on fatty acid chain length. Mean increase of interferon titers of treated relative to control cells (hatched bars, 1 n-&f; open bars, 5 mM).

saturated carbon chain; the results are shown in Table 2. Crotonic acid is the unsaturated analog of n-butyric acid and a likely intermediate in its biological degradation; no enhancing effect was observed when cells were pretreated at concentrations of 5 d. Another possible metabolic intermediate, P-hydroxybutyric acid, was likewise ineffective. A fourfold increase in interferon production was observed with isobutyric acid although it remains undetermined whether this effect was due to the presence of a small amount of n-butyric acid. Also of interest seemed the group of (Y, w-clicarboxylic acids with three to five

TABLE EFFECTS

actd

2

MONOCARBOXYLIC

ACIDS

ON INTERFERON

PRODUCTIONS

Interferon

titer Proportional increase

Concentration (mM) n-Butyric acid Isobutyric acid Crotonic acid P-Hydroxybutyric p-Bromobutyric

acid acid

1 5 5 5 lb

o Treatment of cells and interferon induction were h A concentration of 5 mM was highly toxic.

Number of experiments 7 3 3 2 2 carried

Range (unit&O6 cells) 1,500-15,500 230-2,300 20-200 90-100 80-100

out essentially

as described

Range

Mean

13-37 3-5 0.4-l 0.2-l 0.2-l

29 4