Archives of Virology

Archives of Virology 54, 271--277 (1977)

© by Springer-Verlag 1977

Sensitivity of Various tIuman Lymphoblastoid Cells to the Antiviral and Antieellular Aetivity of Human Leukoeyte Interieron Brief Report By J. HILF]~NI~AUS,It. I)A~I~L and R. JOHA~SE~¢ Behringwerke AG, Marburg/Lahn, Federal Republic of Germany "With 1 Figure Accepted March 17, 1977

Summary Of eight lymphoblastoid cell lines studied five were insensitive to both the anticellular and antiviral activities of human leukocyte interferon, and two were sensitive to both activities. One line could not be fully evaluated since it was not possible to study its sensitivity to the antiviral activity. . Growth inhibition of human cells by h u m a n interferon (HIF) preparations has been extensively studied (e.g., 1, 2, 4, 10, 11, 15, 16, 20). There is some good evidence now that the growth inhibitory ( = anticellular) activity of these preparations is linked to the interferon molecule (12, t5, 20). Depending on the cell lines studied, different results were obtained regarding sensitivity to the antiviral and anticellular activities of interferons. I n some cases the sensitivities to both properties ran in parallel (8), whereas in others the cells were sensitive only to the antiviral property but not to the anticellular one (2, 4, t6). In order to find out whether or not cells of the same type, namely lymphoblastoid cells, revealed comparable sensitivities to both properties of H I F , we tested the antiviral and the anticellular activity of a h u m a n leukocyte interferon (1HIF) preparation in one marmoset and seven h u m a n lymphobtastoid cell lines. The cell lines used in this study are characterized in Table 1. With the exception of the B 9 5 - - 8 line, all lines are of h u m a n origin and, with the exception of the M o l t 4 line, they carry the Epstein Barr virus (EBV) genome. The cells were propagated in I~PMI t640 medium supplemented with 10 per cent fetal calf serum, neomycin, penicillin and streptomycin. No mycoplasmas could be detected in these cell lines b y a biological assay method [performed according to the proee-

272

J. HILFENIIAUS,J~. DAMN!, and R. JOHANNSEN:

dure p u b l i s h e d in the Code of F e d . R e g u l a t i o n s (U.S.A.) § 610.30 (t973)]. Those lines in which E B V capsid antigens (VCA) can be d e m o n s t r a t e d are considered E B V p r o d u c e r lines. The percentage of VCA synthesizing cells of each line was d e t e r m i n e d b y t h e i n d i r e c t immunolluorescence t e c h n i q u e (9) using acetone-fixed cells, the serum of a h e a l t h y donor (anti-VCA titre 1 : 80, t h e d i l u t i o n used 1 : 20) a n d a g o a t a n t i - h u m a n - i m m u n o g l o b u ] i n serum which was c o n j u g a t e d with isothioc y a n a t e (Behringwerke AG, Marburg, F e d . Rep. G e r m a n y ) . The percentage of VCA-positive cells was o b t a i n e d b y c o u n t i n g a t o t a l of 1000 cells/sample. The results shown in Table 1 agree well w i t h the percentages of VCA synthesizing cells p r e v i o u s l y f o u n d b y others (6, 17). H L - A surface antigens on l y m p h o b l a s t o i d cells were d e t e r m i n e d w i t h a slightly modified m i c r o c y t o t o x i c i t y test according to T ~ A S A K I et al. (22) using two penels consisting of 60 or 250 well defined H L A a n t i s e r a for tissue t y p i n g , d e t e c t i n g 26 a n t i g e n species of t h e A, B a n d C locusL The r a b b i t serum used as c o m p l e m e n t source showed nonspecifie c y t o t o x i e

Table 1. Characterization o/the tymphoblastoid cell lines used SFV propagation (log

Cell line ~

Origin

VCA b

H L A antigens

PFUtmt~)

--

7.0

No reproducible results A3, BW17, W35

5.7 d

A3, W 19, B 12, W40, 4a, 4b A 1, 10

7.0

B95-8

(17)

Marmoset leukoeytes transformed b y EBV in vitro

2.7

5.6

I)audi

(14)

Burkitt's l y m p h o m a

0.2--1.0

Diehl I

(6)

H u m a n leukoeytes transformed by EBV

2.5--5.1

Kaplan

(5)

Molt-4

7.3

in vitro

Infect. mononneteosis

0.2--0.6

(18)

Acute lymphatic leukemia

0

P311RI

(13)

Burkitt's lymphoma

3.0--5.5

RPM11788

(21)

Healthy donor

0

SK L 1

(3)

Leukemia.

0

A3, BYVI7 (?), W35 A2, WI9, B7,

14, 4b Unspecific eytolysis

7.1 7.2 6.4

7.3

Numbers in parenthesis refer to references b Percentage of VCA synthesizing cells ¢ Maximum yields of SFV obtained at 9 hours (95-8, Diehl I, Mott-4, P 3 I I R 1, S K L 1) or 24 hours (Kaplan, RPM11788), respectively, in the supernatants of cell cultures infected with SFV at a multiplicity of approx. 5.0 a This result means only a very low SFV propagation as the SFV titre of the supern a t a n t after virus adsorption was 5.0 log PFU/ml. F o r details refer to (12) i The typing reagents were obtained mainly from Behringwerke AG (Marburg, Fed. l~ep. Germany). Some additional sera were generously provided by Dr. J. J. van Rood (Leiden, The Netherlands), .Dr. F. Kissmcyer-Nielsen (Arhus, Denmark), and the NIII serum bank (Bethesda, Md., U.S.A.).

Antiviral and Antieellular Activity of IIuman

Interferon

273

activity with some of the lymphoblastoid cell lines and was therefore adsorbed with 1 × 10 s P 3 H R 1 eells/ml for one hour in an ice water bath. Nevertheless, as shown in Table t, SKL-1 cells were lysed nonspeeifically by most of the a n t i - t t L A sera used. The Daudi line reacted only with some of the sera without a n y correlation to H L A specifieities, thus supporting the results already obtained b y others (19) who had reported on the lack of H L A antigen on the surface of these cells. I H I F was induced by means of Newcastle disease virus in peripheral leukoeyges obtained from heMthy donors, partially purified and tested for antiviral activity as described elsewhere (t 0). Expressed in units of the British research h u m a n interferon standard 69/19A, the 1HIF preparation used had a specific antivirM activity of 1 × 105 units per mg protein. A control preparation, mock interferon (moek-lHIF), was prepared from unindueed peripheral leukoeytes. I t was concentrated and purified in exactly the same way as the 1HIF preparation. Two methods were used to determine the amount of 1HIF necessary for a 50 per cent inhibition of cell growth in comparison to the control, i) 2 × 105 cells/ml were incubated with 1HIF dilutions in Erlenmeyer flasks for five days and final cell concentrations determined by cell count with the trypan blue exclusion assay, ii) 1 × 105 cells/ml were incubated with various 1HIF dilutions in MieroTest plates (Falcon Plastics, Los Angeles, CA, U.S.A.) for four days and then evaluated for eell multiplication b y !4C-TdR uptake as described previously (11). t~or testing the antiviral activity of t H I F in. lymphoblasts, cells in suspension cultures which had reached a density of approx. 1.5 × 106 cells/ml were sedimented b y low speed eentrifugation and resuspended at a similar concentration in complete fresh medium containing various I H I F concentrations. After an overnight incubation cells were again sedimented for removal of 1HIF and, after resuspending them in serum-free RPMI1640 medium, they were infected with Semliki Forest virus (SFV) at a multiplicity of approximately 5 PFU/eell as described elsewhere (12). Maximum SFV titres yielded in the supernatants of infected celt cultures in the absence of interferon treatment are shown in Table 1. I t should be noted that 1.5 hours after virus adsorption SFV titres in the culture supernatants were log 4.5 to log 5.0 P F U / ml. As has been already discussed in detail recently, SFV scarcely propagates in Daudi cells (12). The celt growth curves and the influence of the 1HIF and m o c k - l H I F preparations on cell multiplication are shown in Figure 1. These results indicate t h a t the Daudi line is highly sensitive to the anticellular action of I H I F but not to mock-lHIF, while the Diehl I line is sensitive to 1HIF and, to a lesser extent, to the control preparation as well. These sensitivities of the Diehl I to I H I F and moek~lIIIF resemble those of the P 3 H R 1 on which, we reported in a previous paper (10). 1HIF caused no inhibition or only slight growth inhibition of the marmoset line B 9 5 - - 8 and of the Kaplan, Molt-4 and R P M I 1788 lines. Mock1HIF also showed no effect on the propagation of these cell lines (results not shown). The antieellular activity (on Diehl I and P3I-IR 1 cells) of moek-lI-IIF which lacked any antiviral activity ~ a s stable at p H 2.0 at 4 ° C, but labile to tryptic digestion. Cell multiplication was only inhibited when m o c k - I H I F was present in the cell cultures. After removal of m o e k - l H I F from the cell cultures, cells continued to propagate normally. These properties of the antieellularly active substance of the m o c k - l H I F preparation are comparable to that of 1HIF.

274

J . HII~FE~-i~vS K . DAMM, a n d R . JO~A~NSE~:

The results of virus yield reduction by 1HIF in lymphoblastoid cells are shown in Table 2 (column 5 and 6). In our oppinion a presupposition for a clear antiviral effect of interferon in Shese systems is 100-fold increase of the infectivity titre in ~he control culture, in comparison to that after virus adsorption, and, considering •

•

•

•

O0

O0

•

'-~

oo ~-°°

"O

OOO0

--

H 0

W

~5

I

\'~,

I

|e ~"

I

I

I

I

I

t

o

t t

\

O0

"go

I ~

o

...........i

*--i

m

O0 | !

b

000

C:O

"4"~,¢,k.

I b

R

g m

I

1

I

I o

I

I

I

o

sO L x ILU I SllBa ~o Jaquanu F i g . I. Effeo~ of h u m a n l e u k o c y t e i n t e r f e r o n o n t h e g r o w t h of v a r i o u s l y m p h o b l ~ s ~ o i d cell lines. U n t r e a t e d c o n t r o l cells - , cells ~reated wi~h 1000 u n i t s / m l - - - - - - or 4000 u n i t s / m l of 1 H I F . . . . . , cells t r e a t e d wi~h m o c k - l H I F a t a c o n c e n t r a t i o n e q u i v a l e n t t o 1000 u n i t s / m l . . . . . or 4000 u n i t s / m l of 1 H I F

Antiviral and Anticellular Activity of Human Interferon

275

the experimental error (particularly of SFV titration), a 10-fold reduction of the virus yield in the l l t I F pretreated cell culture in comparison to that of the nontreated control. We therefore conclude that the B95--8, Kaplan, Molt-4, R P M I 1788 and SKL-1 lines are barely sensitive or are insensitive to the antiviral activity of l H I F while the Diem I and P 3 H g l show good sensitivity. I t should be added that mock-lHIF, which is free from antiviral activity in the plaque reduction assay in U cell cultures, also did not inhibit virus propagation in Diem I cells. Table 2. Comparison o] the sensitivqties o] lymphobtastoid cell lines to the antivirat and to the anticellular activity o] human leukocyte inter]eron 50 per cent eM1 growth inhibition a by EBV-

SFV yield reduction (log 1)FU) by pretreatment with 1HIF at a concentration of

Cell line

producer

mock-lHIFb 1HIF units/n~ units/ml

250 units/ml

B 95-8 Daudi Diehl I Kaplan Molt-4 P 3 HI% t I%PMI 1788 SKL- 1

yes yes yes yes no yes no no

> 4000 > 4000 2000 > 4000 >4000 2000 > 4000 >=4000

0.1 0.5 No SFV propagation in Daudi cells 1.9 2.0 0.4 0.7 0.9 1.0 1.2 1.7 0.4 0.6 0.0 0.0

~4000 15 500 > 4000 >4000 250 > 4000 2000

1000 units/ml

a Determined by the 14C-TdR uptake method after a five day incubation period b Concentrations of mock-lHIF given in units of those of an equivalent 1HIF preparation Comparison of SFV yield reduction in lymphoblastoid cells by 1HIF with the growth inhibitory effect on these cells (Table 2) indicates that those cell lines which are not sensitive to the anticellular activity of 1HIF axe also poorly sensitive to the antiviral action. However, the situation becomes more complicated in the case of those cell lines which are sensitive to the anticellular activity of 1HIF. i) The Daudi line, which is very sensitive to the anticellular activity of l t I I F but not to that of moek-ltiIF, does not propagate the challenge virus SFV and therefore the antiviral activity could not be tested in this line. The poor propagation of SFV in Daudi cells agrees with the results obtained b y ADA~IS et al. (1) who reported on the propagation of VSV in Daudi cells. In their experiments these cells (5 × 105 eells/ml) infected with VSV at a multiplicity of 0.06 PFU/eell (by a rough estimate this corresponds to a residual titre of log 2.0 PFU/ml in the culture supernatant after virus adsorption and one cell wash) yielded only log 3.2 P F U / m l 48 hours p.i. if) The Diem I and P 3 H l g l lines, which are sensitive to the antiviral activity of IHIF, are not only sensitive to the antice]lular activity of I H I F but also to that of moek-lHIF. This is comparable with the results obtained by G R ~ T and co-workers (7) who described some "proliferation inhibitory activity" in the supernatants of " u n s t i m u ] a t e d " peripheral human leukocytes. If we take these limitations into account we can conclude that none of the three cell lines sensitive to the anticellular activity of 1HIF was proven to be insensitive to the antiviral activity of 1HIF. I n fact two of them could be shown to be sensitive

276

J. HILFENIIAUS,

H. DASh,, and I%. JOHANNSEN:

to the a n t i v i r a l a c t i v i t y . Our results agree with those o b t a i n e d b y ADAMS et al. (1) who showed the h u m a n l y m p h o b l a s t o i d cell line R a j i to be r a t h e r insensitive to b o t h p r o p e r t i e s of 1HIF, a n d even. m o r e w i t h those of GRESS~R et al. (8) who described m u r i n e l e u k e m i a cell lines which were either sentitive or insensitive to b o t h p r o p e r t i e s of m u r i n e interferon. However, our results are in less g o o d agreem e n t w i t h t h e results of others (2, 4, 16) who s t u d i e d t h e a n t i v i r a l a n d a n t i c e l l u l a r a c t i v i t y of l H I F in various h u m a n cell lines. This could be due to t h e fact t h a t a correlation of t h e sensitivities to b o t h interferon p r o p e r t i e s o n l y exists for cell lines of t h e same celt t y p e or even o n l y for those d e r i v e d from white blood cells.

Aeknowled 9ements We are indebted to Dr. H. zur Hausen (Inst. f. Klin. Virol., Erlangen, Fed. Rep. Germany) and to Dr. F. Seiler (Behringwerke AG, Marburg, Fed. t~ep. Germany) for the supply of tymphoblastoid celt lines. The technical assistance of h~[iss E. Hfihn, Miss C. Teuter and Mrs. H. Thierfdder is gratefully acknowledged.

References I. ADApts, A., STRANDER, I~., CANTELL, K. : Sensitivity of Epstein-Barr virus transformed human lymphoid cell lines to interferon. J. geE. Virol. 28, 207--217 (1975). 2. BO~ECK~, L., FUCHSB~I~GEI% RL, HA;T~C~:i, V.: Electrophore~ic profiles and activities of human interferon in heterologous cells. Intervirology 3,369--377 (1974). 3. CLA~aKSON, B., STRISS, A., DE I-I~i~vE~r, E. : Continuous culture of seven new lines (SKL-I to 7) from patients with acute leukemia. Cancer 20, 926--947 (1967). 4. DAHL, I~., DEGI~, M. : Human interferon and cell growth inhibition. I. Inhibitory effect of human interferon on the growth rate of cultured human cells. Acta path. microbiol, scand. Sect. B 84, 285----292 (1976). 5. DIEIIL, V., HENLE, G., HELLE, W., KOIIN, G. : Demonstration of herpes group virus in cultures of peripheral leukocytes from patients with infectious mononucleosis. J. Virol. 2, 663--669 (1968). 6. DIEHL, V., WOLF, H., ScnULTE-HOLTIIAUSEI% Hi., ZUl% HAUSEN, H. : l~e-exposure of human lymphoblastoid cell lines to Epstein-Barr virus. Int. J. Cancer I0, 641 to 651 (1972). 7. GREEN, H. A., COOPEI~BAND, S. R., RUTSTEI1N, J. A., KIBRIOK, S.: Inhibition of target cell proliferation by supernatants from cultures of human peripheral lymphocytes. J. Immunol. 105, 48--54 (1970). 8. GI~ESSEka, I., BA:NDU, M.-T., BJ~ou:rY-BoY~, D. : Interferon and cell division. IX. Interferon-resistent L 1210 cells: Characteristics and origin. J. nat. Cancer Inst. 52, 553--559 (1974). 9. HENLE, G., HELLE, W. : Immunofluorescence in cells derived from Burkitt's lymphoma. J. Bacteriol. 91, 1248---1256 (1966). 1O. HIL~ENI{AUS, J., KAI%GES, H. E. : Growth inhibition of human lymphoblastoid cells by interferon preparations, obtained from human leukocytes. Z. Naturforsch. 29 c,

618--622 (1974). ] l. HILFENttA~'S, J., DAM~, I~., KARGES, H. E., MANTI~ttEY,K. F. : Growth inhibition of human Iymphoblastoid Daudi cells in vitro b y interferon preparations. Arch. Virol. 51, 87--97 (1976). t2. HILFENHA~-S, J'. : Propagation of Semliki Forest virus in various h u m a n lymphoblastoid cell lines. J. geE. Virol. 33, 539---542 (1976). 13. HINU/CIA, Y., KONN, M., YA2¢IAGUCIII, Y., WUDARSKI, D. J., BLAKESLEE, J. R., G~AOE, J. T. : Immunofluorescence and herpes-type virus particles in the P 3I~R i Burkitt ]ymphoma cell line. J. Virol. I, 104,5--1051 (1967).

A n t i v i r a l and Antieellular A c t i v i t y of H u m a n I n t e r f e r o n

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14. KLEIN, E., KLEIN, G., NADKARNI, J. S., NADKARNI, J. J., WIGZELL, H., CLIFFORD, P. : Surface IgM k a p p a specificity on a B u r k i t t l y m p h o m a cell i n vivo and d e r i v e d culture lines. Cancer Res. 28, 1300--1310 (1968). 15. KNIGHT, E., JR. : A n t i v i r a l a n d cell growth i n h i b i t o r y activities reside in the same glycoprotein of h u m a n fibroblast interferon. N a t u r e (Lond.) 2{}2, 302--303 (1976). 16. KUWATA, T., FUSE, A., MORIGANA,N. : Effects of interferon on cell and virus g r o w t h in t r a n s f o r m e d h u m a n ceil lines. J. gen. Virol. SS, 7 - - 1 5 (1976). 17. MILLER, G., S~IOFE, T., LISCO, H., STITT, D., LIP~A~, M.: E p s t e i n - B a r r virus: Transformation, c y t o p a t h i c changes, and viral antigens in squirrel m o n k e y and m a r m o s e t leukocytes. Proc. nat. Acad. Sci. (Wash.) 69, 383--387 (1972). 18. MII~OWADA, J., OHNUMA, T., MOORE, G. E . : R o s e t t e - f o r m i n g h u m a n l y m p h o i d cell lines. I. E s t a b l i s h m e n t and evidence for origin of t h y m u s - d e r i v e d l y m p h o c y t e s . J. nat. Cancer Inst. 49, 891--895 (1972). 19. POULIX, M. D., FERRONE, S., PELLEGRINO, M. A., SEVIER, D. H., OK, S. K., REISFELD, R. A. : Association of H L - A antigens and ~ 2-micro-globulin: Concepts and questions. Transplant. R e v . 21, 106--125 (1974). 20. STEWART I I , W. E., GRESSER, I., TOVEY, M. G., BAIqDU, M.-T., LE GOFF, S.: I d e n t i f i c a t i o n of t h e cell multiplication i n h i b i t o r y factors in interferon preparations. N a t u r e (Lond.) 262, 302--303 (1976). 21. TAKADA, A., TAKADA, Y., MINOWADA, J . : I m m u n o l o g i c a l functions of h u m a n Tl y m p h o i d cell line (MOLT). J. exp. Med. 140, 538 548 (1974). 22. TERASAKI, P. I., MCCLELLAND, J., PARK, M. S., McCuRDY, B.: Microdroplet l y m p h o c y t e c y t o t o x i c i t y test. I n : RAY, J. G., HARE, D. B., PEDERSEN, P. D., KAYHOL, D. E. (eds.), Manual of Tissue T y p i n g Techniques, D H E W Publ. No. (NIH) 75--545, p. 6 7 - - 7 4 (1974). A u t h o r s ' address: Dr. J. HILFENttAUS, Behringwerke AG, D-3550 M a r b u r g / L a h n , Federal R e p u b l i c of G e r m a n y . R e c e i v e d J a n u a r y 17, 1977

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