Interferon-Carbohydrate Interaction - ACS Symposium Series (ACS

22 Interferon-Carbohydrate Interaction 1

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HELMUT ANKEL, FRANÇOISE BESANCON, and CHITA KRISHNAMURTI Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226

Previous investigations suggest that mouse fibroblast interferon interacts with carbohydrate-containing cell membrane constituents. Its antiviral action is blocked by certain plant lectins, such as those from Phaeseolus vulgaris (PHA, ref. 1) or the nontoxic agglutinin from Abrus precatorius (2), and after preincubation with gangliosides (3,4). Gangliosides covalently attached to Sepharose avidly bind mouse fibroblast interferon, which is reversed in the presence of N-acetylneuraminyl lactose, the trisaccharide common to many gangliosides (4). Furthermore, preincubation of SV/ALN cells with gangliosides under conditions that lead to incorporation into the cell membrane of these cells, increases their sensitivity to the antiviral action of mouse fibroblast interferon as described by Vengris, Reynolds, Hollenberg and Pitha (5). In this communication we extend our earlier observations, which primarily dealt with the antiviral action of mouse fibroblast interferon, to its antigrowth activity and to antiviral and antigrowth activities of mouse T-cell interferon. We will show that inhibition by common gangliosides is restricted to both activities of fibroblast interferon alone. T-cell interferon, although its biological activities are analogous to those of fibroblast interferon, neither binds to nor is inhibited by these glycolipids. Furthermore we demonstrate that mouse leukemia L-1210 cells that were selected for resistance to fibroblast interferon (6), respond equally well to T-cell interferon as the parent cells which are responsive to both interferons. Materials and Methods CelIs and Virus. Encephalomyocardîtîs virus (EMC) and mouse L929 fibroblasts were obtained from Dr. Sidney Grossberg. L929 cells were routinely propagated in MEM containing ]0% fetal bovine serum (Gibco). Fibroblast interferon-sensίtive and resistant Current address: Laboratoire de Biochimie Physique, I.N.R.A. Université Paris-Sud, 91 ^05 Orsay, France 0-8412-0556-6/80/ 47-128-391 $5.00/ 0 © 1980 American Chemical Society Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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mouse l e u k e m i a L - 1 2 1 0 c e l l s ( L - 1 2 1 OS a n d L-121 OR) were p r o v i d e d by D r . Ion G r e s s e r and were c u l t u r e d i n RPMI 2310 medium ( G i b c o ) s u p p l e m e n t e d w i t h 5% f e t a l b o v i n e s e r u m . Reagents. P a r t i a l l y p u r i f i e d mouse f i b r o b l a s t interferon was s u p p l i e d by D r . K u r t P a u c k e r and had a s p e c i f i c a c t i v i t y o f l.k χ 1 0 NIH R e f e r e n c e U n i t s p e r mg p r o t e i n ( l U / m g ) . Mouse T c e l 1 i n t e r f e r o n was p r o d u c e d i n PHA s t i m u l a t e d mouse s p l e e n cells i n c u l t u r e and p u r i f i e d by D r . E r n e s t o F a l c o f f ( 7 ^ 8 ) . Its s p e c i f i c a c t i v i t y was 1.6 χ 1 0 lU/mg. Mono- and o l i g o s a c c h a r i d e s , mixed b o v i n e b r a i n g a n g l i o s i d e s a n d p o l y l y s i n e (MW 3 0 , 0 0 0 ) were from S i g m a , C N B r - a c t i v a t e d S e p h a r o s e from P h a r m a c i a , and i n d i v i dual g a n g l i o s i d e s from S u p e l c o . G a n g l i o s i d e s G^3 a n d G ^ 2 were k i n d l y p r o v i d e d by D r . Subhash B a s u . Individual gangliosides are d e s i g n a t e d a c c o r d i n g t o S v e n n e r h o l m (9). T h e i r p u r i t i e s were a n a l y s e d by TLC ( s e e b e l o w ) . G ^ i , G ^ a n d G ^ 3 showed s i n g l e spots a f t e r exposure to r e s o r c i n o l spray o r iodine vapor. G was s l i g h t l y c o n t a m i n a t e d w i t h a r e s o r c î n o l - p o s î t i v e s p o t w i t h t h e m o b i l i t y o f G M , & T l b showed o n e a d d i t i o n a l s p o t w h i c h moved i d e n t i c a l l y to G p i ^ . B o t h c o n t a m i n a n t s were p r e s e n t i n amounts o f 10% o r l e s s a s j u d g e d from t h e r e l a t i v e i n t e n s i t i e s o f t h e s p o t s . The mixed g a n g l i o s i d e f r a c t i o n from mouse b r a i n was p r e p a r e d a c c o r d i n g t o F o l c h e_t a k (10) as d e s c r i b e d by B r u n n g r a b e r e t a l . (11). G a n g l i o s i d e a f f i n i t y columns were p r e p a r e d a s p r e v i o u s l y d e s c r i b e d (k). The procedure i n v o l v e d c o u p l i n g o f p o l y l y s i n e to C N B r - a c t i v a t e d Sepharose (12), f o l l o w e d by t h e a t t a c h m e n t o f mixed b o v i n e b r a i n g a n g l i o s i d e s w i t h c a r b o d î î m i d e ( 1 3 ) . 7

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A n t i v i r a l Assay. A n t i v i r a l a c t i v i t y was d e t e r m i n e d i n mouse cells. A p p r o x i m a t e l y 1 0 c e l l s i n 1 ml MEM p l u s 10% f e t a l b o v i n e serum were s e e d e d i n t o 16 mm w e l l s o f M u l t i W e l l t i s s u e c u l t u r e p l a t e s ( F a l c o n ) and kept a t 3 7 ° i n a C 0 i n c u b a t o r . The f o l l o w i n g day medium was removed and c e l l s were i n c u b a t e d w i t h a p p r o p r i a t e d i l u t i o n s o f i n t e r f e r o n i n s e r u m - f r e e MEM c o n t a i n i n g 50 y g / m l b o v i n e serum a l b u m i n ( 0 . 5 m l / w e l l ) . Where i n d i c a t e d , i n t e r f e r o n s o l u t i o n s were p r e i n c u b a t e d w i t h g l y c o l i p i d s o r c a r b o h y d r a t e s a t 3 7 ° f o r 30 m i n u t e s p r i o r t o a d d i t i o n t o t h e c e l l monolayers. C o n t r o l c e l l s were i n c u b a t e d u n d e r i d e n t i c a l c o n d i t i o n s , but i n t h e a b s e n c e o f i n t e r f e r o n . A f t e r 2h h o u r s a t 3 7 ° , medium was removed a n d t h e c e l l s were i n f e c t e d w i t h EMC a t a m u l t i p l i c i t y o f i n f e c t i o n o f 0 . 1 , a d d i n g 0 . 5 ml o f an a p p r o p r i a t e v i r u s s u s p e n s i o n i n MEM-2% f e t a l b o v i n e serum t o e a c h w e l l . A f t e r 1 hour a t 3 7 ° medium w i t h n o n - a d s o r b e d v i r u s was removed and t h e c e l l s were i n c u b a t e d i n 0 . 5 ml f r e s h medium p l u s 2% f e t a l b o v i n e serum f o r 16 t o 17 h o u r s a t 3 7 ° . T h e y were t h e n p l a c e d a t - 8 0 ° f o r a t l e a s t 30 m i n u t e s , t h e n thawed and v i r u s y i e l d i n e a c h w e l l was d e t e r m i n e d by h e m a g g l u t i n a t i o n o f human r e d b l o o d c e l l s o f t y p e 0 in s e r i a l t w o - f o l d d i l u t i o n s o f the v i r u s suspensions ( 1 4 ) . EMC t i t e r s a r e e x p r e s s e d as t h e r e c i p r o c a l s o f t h e h i g h e s t d i l u t i o n s t h a t s t i l l showed h e m a g g l u t i n a t i o n . The a c c u r a c y o f t h i s assay i s

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about ± one d i l u t i o n . When t h e e f f e c t s o f f i b r o b l a s t and T - c e l l i n t e r f e r o n s a n d t h o s e o f d i f f e r e n t g l y c o s i d e s on t h e s e i n t e r f e r ons were c o m p a r e d , e x p e r i m e n t s were p e r f o r m e d s i m u l t a n e o u s l y u s i n g t h e same b a t c h o f L - c e l l s u n d e r i d e n t i c a l c o n d i t i o n s . T h i s was n e c e s s a r y s i n c e i n t e r f e r o n s e n s i t i v i t y a n d v i r a l y i e l d were somewhat v a r i a b l e f r o m c u l t u r e t o c u l t u r e . Concentrations of i n t e r f e r o n used i n i n d i v i d u a l experiments r e f e r to a p p r o p r i a t e d i l u t i o n s o f t h e o r i g i n a l s o l u t i o n s , whose i n t e r f e r o n t i t e r s were d e t e r m i n e d by c o m p a r i n g t h e i r a n t i v i r a l a c t i v i t i e s t o t h o s e o f NIH s t a n d a r d G 0 0 2 - 4 9 - 5 1 1 , o n e l U / m l r e f e r r i n g t o an i n t e r f e r o n c o n c e n t r a t i o n t h a t r e s u l t s i n 50% i n h i b i t i o n o f v i r a l y i e l d a s compared t o t h e s t a n d a r d . Growth S t u d i e s . L - 1 2 1 0 R a n d L - 1 2 1 0 S c e l l s were grown w i t h o u t a g i t a t i o n i n p l a s t i c v i a l s i n RPMI medium c o n t a i n i n g 5% f e t a l b o v i n e serum a t 3 7 ° i n a C 0 i n c u b a t o r , u s i n g between 0 . 3 and 1 ml c u l t u r e medium i n d i f f e r e n t e x p e r i m e n t s . C e l l c o u n t s were done in a C o u l t e r c o u n t e r . When L - 1 2 1 0 R and L-121 OS c e l l s were comp a r e d , c u l t u r e s were i n v e s t i g a t e d s i m u l t a n e o u s l y and u n d e r i d e n t i cal c o n d i t i o n s . 2

Column C h r o m a t o g r a p h y . S e p h a r o s e beads c o n t a i n i n g c o v a l e n t l y l i n k e d g a n g l i o s i d e s ( 0 . 2 ml p a c k e d volume) w e r e p l a c e d i n t o a p a s t e u r p i p e t t e c o n t a i n i n g a s m a l l amount o f g l a s s w o o l . Columns were washed w i t h MEM c o n t a i n i n g 50 y g / m l b o v i n e serum a l b u m i n (3 m l ) . I n t e r f e r o n s o l u t i o n s i n M E M - a l b u m i n (1 ml) were p l a c e d on the c o l u m n s , w h i c h were e l u t e d w i t h M E M - a l b u m i n a t a f l o w r a t e o f no more t h a n o n e d r o p p e r m i n u t e . F r a c t i o n s o f 1 ml were c o l l e c t ed and i n t e r f e r o n t i t e r s d e t e r m i n e d i n e a c h f r a c t i o n a f t e r s e r i a l two-fold d i l u t i o n . Columns o n t o w h i c h mouse f i b r o b l a s t i n t e r f e r o n had been l o a d e d , were e l u t e d w i t h M E M - a l b u m i n f i r s t , t h e n w i t h 0 . 0 7 M N - a c e t y l n e u r a m i n y l l a c t o s e a t pH 2. Other A n a l y t i c a l Procedures. T h i n l a y e r c h r o m a t o g r a p h y was c a r r i e d o u t w i t h s i l i c a g e l p l a t e s G60 ( M e r c k ) , u s i n g e i t h e r chloroform-methanol-water (65:^5:9) o r n-propanol-0.2% C a C l in H 0 (80:20). T h e p l a t e s were d e v e l o p e d w i t h r e s o r c i n o l s p r a y (15). S i a l i c a c i d was d e t e r m i n e d w i t h t h î o b a r b î t u r i c acid after h y d r o l y s i s i n 0.1 s u l f u r i c a c i d ( 1 6 ) . 2

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t o be s p e c i e s - s p e c i f i c , we i n v e s t i f r a c t i o n f r o m mouse b r a i n was more a c t i v i t y o f mouse f i b r o b l a s t i n t e r heterologous brain e x t r a c t s . As g a n g l i o s i d e s were a l m o s t as p o t e n t


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2048 512 Figure 1. Effects of mouse (A.) and bovine (%) brain gangliosides on anti viral activity of mouse fibroblast inter feron

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Interferon solutions in MEM (30 lU/mL) were preincubated with the indicated gan glioside concentrations at 37°C for 30 min before addition to the L-cell monolayer. Antiviral assays are described in Materials and Methods. Y : EMC titer in the absence of interferon. This titer was unchanged in the presence of both ganglioside preparations up to a concentration corresponding to 100 μ Μ sialic acid.

0 1.25 2.5 5 10 20 GANGLIOSIDE CONCENTRATION [ μ Μ SIALIC ACID]

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Figure 2. Effects of individual glyco lipids on antiviral activity of mouse fibro blast interferon Experimental conditions as in Figure 1. O. Globoside; · , G ; Δ , GM1; A , G ; •, G ; •. G^ . V: EMC titer in the absence of interferon. This titer was unchanged in the presence of each individual glycolipid up to a concentration of 100 μΜ. MS

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în i n h i b i t i n g a n t i v i r a l a c t i v i t y as t h o s e d e r i v e d f r o m m u r i n e brain. The l i n e s e x t r a p o l a t e t o g a n g l i o s i d e c o n c e n t r a t i o n s c o r r e s p o n d i n g t o 12 and 18 yM s i a l i c a c i d , r e s p e c t i v e l y , f o r complete inhibition of a n t i v i r a l a c t i v i t y . We d o u b t t h a t t h i s a p p a r e n t 1.5 f o l d d i f f e r e n c e i s s i g n i f i c a n t , s i n c e t h e v i r a l a s s a y c a n n o t be c a r r i e d o u t w i t h enough a c c u r a c y t o s u p p o r t d i f f e r e n c e s o f l e s s t h a n a f a c t o r o f 2. B o t h p r e p a r a t i o n s had v e r y s i m i l a r p a t t e r n s o f m a j o r g a n g l i o s i d e s when a n a l y z e d by T L C , i n a c c o r d a n c e w i t h o b s e r v a t i o n s by o t h e r s , who f i n d s i m i l a r r a t i o s o f g a n g l i o s i d e s G ^ ! , Gnia» G p i b and G j i ^ i n b r a i n e x t r a c t s f r o m b o v i n e and murine o r i g i n (17). In e a r l i e r work we u s e d p r e i n c u b a t i o n o f S e p h a r o s e - b o u n d mouse f i b r o b l a s t i n t e r f e r o n w i t h s o l u t i o n s o f i n d i v i d u a l g a n g l i o s i d e s t o d e m o n s t r a t e t h e i r e f f e c t on a n t i v i r a l a c t i v i t y ( 3 , ^ 0 . C o m p a r i s o n o f p o t e n c y o f i n h i b i t i o n by i n d i v i d u a l g l y c o l i p i d s u n d e r t h e s e s e m i - q u a n t i t a t i v e c o n d i t i o n s i n d i c a t e d t h a t G^2 and G j i b were e q u a l l y good i n h i b i t o r s , and t h a t G n i and G ^ i were somewhat l e s s i n h i b i t o r y . G^3 was o n l y s l i g h t l y i n h i b i t o r y , whereas g a n g l i o - t r i h e x a o s y l c e r a m i d e , g l o b o - t r i h e x a o s y l ceramide and g l o b o s i d e had no e f f e c t . S i n c e g a n g l i o s i d e G^3 and g a n g l i o t r i a o s y l c e r a m î d e were much l e s s i n h i b i t o r y t h a n G M o r d i d n o t i n h i b i t at a l l , i t appeared that terminal N - a c e t y l g a l a c t o s a m i n y l and ( o r ) N - a c e t y l n e u r a m i n y l r e s i d u e s a r e i m p o r t a n t c o n s t i t u e n t s for i n h i b i t i o n of a n t i v i r a l a c t i v i t y . In o r d e r t o o b t a i n more q u a n t i t a t i v e d a t a on t h e r e l a t i v e I n h i b i t o r y p o t e n c i e s o f i n d i v i d u a l g a n g l i o s i d e s , we s u b s e q u e n t l y p r e l n c u b a t e d u n d e r i v a t i z e d mouse f i b r o b l a s t I n t e r f e r o n w i t h g a n g l i o s i d e s o l u t i o n s p r i o r to the a d d i t i o n to the t a r g e t c e l l s . The d a t a shown i n F i g u r e 2 c o r r o b o r a t e o u r e a r l i e r o b s e r v a t i o n s , i n d i c a t i n g t h a t under t h e s e c o n d i t i o n s i n d i v i d u a l g l y c o l i p i d s w i l l cause complete I n h i b i t i o n o f a n t i v i r a l a c t i v i t y o f f i b r o b l a s t I n t e r f e r o n i n t h e f o l l o w i n g o r d e r : G j i ^ and G ^ > G p i > G^i > M3 > g l o b o s i d e , r e q u i r i n g i n d i v i d u a l c o n c e n t r a t i o n s o f ]k 30, hS 100 and 1000 yM f o r c o m p l e t e r e v e r s a l o f t h e a n t i v i r a l e f f e c t . a

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S i n c e t h e c e r a m i d e p o r t i o n s o f more and l e s s I n h i b i t o r y g l y c o l i p i d s are very s i m i l a r , d i f f e r e n t i a l i n h i b i t i o n o f a n t i v i r a l a c t i v i t y o f mouse f i b r o b l a s t i n t e r f e r o n must be r e l a t e d t o t h e i r carbohydrate side chains. We t h e r e f o r e a s s a y e d a n t i v i r a l a c t i v i t y in t h e p r e s e n c e o f v a r i o u s s a c c h a r i d e s c o n t a i n e d i n g a n g l i o s i d e s . As seen i n F i g u r e 3, b o t h N - a c e t y 1 n e u r a m i n y l l a c t o s e and N - a c e t y l neuramlnic a c i d i n h i b i t e d a n t i v i r a l a c t i v i t y , r e q u i r i n g a p p r o x i m a t e l y e q u a l c o n c e n t r a t i o n s t o o b t a i n c o m p l e t e I n h i b i t i o n (60 mM). H o w e v e r , In c o m p a r i s o n t o G M , 6 0 0 - f o l d h i g h e r c o n c e n t r a t i o n s o f t h e s e s u g a r s had t o be e m p l o y e d t o y i e l d c o m p l e t e i n h i b i t i o n o f antiviral activity. N - g l y c o l y l n e u r a m l n l c a c i d and t h e $ - m e t h y 1 3


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Figure 3.

Effects of mono- and oligosaccharides on antiviral activity of mouse fibroblast interferon

Experimental conditions as in Figure 1. M, Lactose; Δ , N-glycolylneuraminic acid; A. neuraminic acid β-methyl glycoside; O , N-acetylneuraminic acid; · , N-acetylneuraminyl lactose. T: EMC titer in the absence of interferon. This titer was unchanged in the presence of each saccharide up to a concentration of 100 mM.

Figure 4. Binding of mouse fibroblast interferon to Sepharose-ganglioside colums and elution with N-acetylneuraminyl lactose One mL interferon solution (2 Χ 10 IU) in MEM plus 50 y,glmL bovine serum albumin was loaded onto a small column containing 0.2 mL of the Sepharose-ganglioside adduct as described in Materials and Methods. The column was first eluted with MEM-albumin alone. At arrow, elution was continued with a solution of 0.07M N-acetylneuraminyl lac tose in MEM-albumin at pH 2. Antiviral activity in each fraction was determined as described in Materials and Methods. A small amount of the antiviral activity (7% ) passed the column unretarded; the remaining por tion (89% of that applied) was eluted with N-acetylneuraminyl lactose. 3

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g l y c o s i d e o f n e u r a m i n i c a c i d a l s o were i n h i b i t o r y , y e t c o n c e n t r a t i o n s a p p r o x i m a t e l y t h r e e times h i g h e r than those o f the above s a c c h a r i d e s r e s u l t e d in comparable i n h i b i t i o n . In v i e w o f t h e fact that a l l gangliosides contain substituted l a c t o s y l residues i t i s i n t e r e s t i n g t h a t l a c t o s e had no e f f e c t on a n t i v i r a l a c t i v i t y up t o c o n c e n t r a t i o n s o f 100 mM. O t h e r s u g a r s t h a t had l i t t l e o r no e f f e c t a t c o m p a r a b l e c o n c e n t r a t i o n s w e r e : N - a c e t y l g l u c o s a m î n e , N-acetylgalactosamine, mannose, g a l a c t o s e and L - f u c o s e . It i s s u r p r i s i n g t h a t t h e t r i s a c c h a r i d e N - a c e t y l n e u r a m i n y l l a c t o s e was much l e s s p o t e n t i n i n h i b i t i n g a n t i v i r a l a c t i v i t y than the c o r r e s p o n d i n g g l y c o l i p i d G M . T h i s might i n d i c a t e t h a t e i t h e r t h e b i n d i n g s i t e o f mouse f i b r o b l a s t i n t e r f e r o n on G M i n c l u d e s p a r t o f t h e l i p i d p o r t i o n as w e l l , o r t h a t a r r a n g e m e n t o f c a r b o h y d r a t e c h a i n s in g a n g l i o s i d e m i c e l l e s f a v o r s a conformation w h i c h a l l o w s f o r a much t i g h t e r f i t o f i n t e r f e r o n . The f r e e t r i s a c c h a r i d e i n s o l u t i o n , on t h e o t h e r h a n d , m i g h t assume any numb e r o f c o n f o r m a t i o n s , o f w h i c h o n l y one o r v e r y few a r e f a v o r a b l e to i n t e r f e r o n b i n d i n g . 3

3

T h a t i n h i b i t i o n o f a n t i v i r a l a c t i o n i s due t o b i n d i n g , and t h a t t h i s i n v o l v e s t h e c a r b o h y d r a t e s i d e c h a i n s on t h e g a n g l i o s i d e m o l e c u l e , was c l e a r l y i n d i c a t e d by t h e b e h a v i o r o f fibroblast i n t e r f e r o n on a f f i n i t y columns c o n t a i n i n g c o v a l e n t l y bound g a n g l i osides. As seen i n F i g u r e k when mouse f i b r o b l a s t i n t e r f e r o n was p l a c e d on s u c h a c o l u m n , l e s s t h a n 10% o f t h e a n t i v i r a l a c t i v i t y passed through u n r e t a r d e d . The r e m a i n i n g a n t i v i r a l a c t i v i t y was q u a n t i t a t i v e l y e l u t e d w i t h 70 mM s o l u t i o n s o f N - a c e t y l n e u r a m i n y l l a c t o s e a t pH 2. It s h o u l d be n o t e d t h a t t h i s c o n c e n t r a t i o n o f the t r i s a c c h a r i d e a l s o c o m p l e t e l y r e v e r s e d the a n t i v i r a l effect, as i n d i c a t e d i n F i g u r e 3. 9

Effect of Gangliosides Interferon.

on A n t i g r o w t h A c t i v i t y o f

Fibroblast

S i n c e i t has been e s t a b l i s h e d t h a t a n t i v i r a l and a n t i g r o w t h a c t i v i t i e s o f mouse f i b r o b l a s t i n t e r f e r o n r e s i d e i n t h e same m o l e cules (18), one w o u l d e x p e c t t h a t g a n g l i o s i d e s w o u l d I n h i b i t b o t h a c t i v i t i e s In a s i m i l a r f a s h i o n . To I n v e s t i g a t e t h e e f f e c t o f g a n g l i o s i d e s on g r o w t h i n h i b i t i o n , we used mouse l e u k e m i a L - 1 2 1 0 c e l l s , w h i c h grow more r a p i d l y t h a n mouse L - c e l l s . This cell line i s o f a d d i t i o n a l i n t e r e s t s i n c e G r e s s e r , Bandu and B r o u t y - B o y e have I s o l a t e d a s u b l i n e ( L - l 2 1 OR) by c o n t i n u o u s g r o w t h In t h e p r e s e n c e o f mouse f i b r o b l a s t I n t e r f e r o n , w h i c h i s r e s i s t a n t t o i t s a n t i v i r a l and a n t i g r o w t h a c t i v i t i e s ( 6 ) . When g r o w t h o f i n t e r f e r o n - s e n s î t î v e L - 1 2 1 0 c e l l s ( L - l 2 1 O S T was f o l l o w e d f o r k d a y s , t h e number o f c e l l s In c o n t r o l c u l t u r e s was t h r e e t i m e s h i g h e r t h a n t h a t In c u l t u r e s w h i c h c o n t a i n e d mouse f i b r o b l a s t Interferon (Figure 5). Although a d d i t i o n of gangliosides alone Inhibited g r o w t h t o some e x t e n t , t h e e f f e c t o f f i b r o b l a s t i n t e r f e r o n In t h e p r e s e n c e o f g a n g l i o s i d e s was l a r g e l y r e v e r s e d and t h e c e l l number In t h e s e c u l t u r e s a p p r o a c h e d t h a t o f c u l t u r e s grown In t h e


398

C E L L

S U R F A C E


Figure 5. Effect of bovine brain ganglio sides on growth inhibition of L-l 21 OS cells by mouse fibroblast interferon Cells were grown in RPMI medium plus 5% fetal bovine serum in 1 mL total volume as described in Materials and Methods. At 24hr intervals the cells were counted in a Coul ter counter. Control cells; · , cells from cul tures containing 1000 lU/mL mouse fibro blast interferon; •, cells from cultures containing bovine brain gangliosides at a concentration corresponding to 35 μΜ sialic acid; W, cells from cultures containing both interferon (1000 IU/mL) and gangliosides (35 μΜ sialic acid).

1 2

3

4

TIME OF CULTURE [DAYS]

Figure 6. Effects of mousefibroblastin terferon L-1210S and L-1210R cells in the presence of increasing ganglioside concentrations Cells were seeded at an original cell density of 8 X 10'> L-1210S cells/mL and 7 X 70* L-l21 OR cells/mL in 0.4 mL total volume. Cells were cultured as in Figure 5 and counted after three days of growth. O, Cells grown in the presence of 1000 IU/mL mouse fibroblast interferon; · , those grown in its absence under identical conditions. Top, L-1210R cells; bottom, L-1210S cells.

0

20

40

60 80

GANGLIOSIDE CONCENTRATION [ μ Μ SIALIC ACID]


22.

A N K E L

E T

Interferon-

A L .

Carbohydrate

Interaction

399

presence o f g a n g l i o s i d e s alone. S i n c e t h e g r o w t h - î n h i b î t o r y e f f e c t o f g a n g l i o s i d e s was much l e s s p r o n o u n c e d a f t e r 3 days o f c u l t u r e , we i n v e s t i g a t e d t h e e f fect o fdifferent ganglioside c o n c e n t r a t i o n s on a n t i g r o w t h a c t i v i t y by c o u n t i n g t h e c e l l s a f t e r 3 d a y s . A comparison o f t h e e f f e c t s o f g a n g l i o s i d e s on a n t i g r o w t h a c t i v i t y o f i n t e r f e r o n i n b o t h L-1210R and L - l 2 1 OS c e l l s i s shown i n F i g u r e 6. As e x p e c t e d f r o m t h e o r i g i n a l o b s e r v a t i o n s by G r e s s e r est aj_. (6) t h e L-121 OR cells were n o t i n h i b i t e d by f i b r o b l a s t i n t e r f e r o n and a d d i t i o n o f g a n g l i o s i d e s had l i t t l e e f f e c t , b o t h i n t h e a b s e n c e and i n t h e presence o f i n t e r f e r o n . On t h e o t h e r h a n d , i n t h e L-121 OS c u l t u r e s i n t e r f e r o n p r o d u c e d a p p r o x i m a t e l y 50% r e d u c t i o n i n c e l l number. The c e l l n u m b e r , h o w e v e r , p r o g r e s s i v e l y i n c r e a s e d w i t h increasing concentrations o fgangliosides. Complete r e v e r s a l o f t h e i n t e r f e r o n e f f e c t was o b s e r v e d a t a g a n g l i o s i d e concentration c o r r e s p o n d i n g t o 70 μΜ s i a l i c a c i d . T h i s compares t o a c o n c e n t r a t i o n o f 20 μΜ f o r c o m p l e t e r e v e r s a l o f t h e a n t i v i r a l e f f e c t i n t h e mouse L - c e l l s y s t e m ( F i g u r e 1 and 7). Effects

o f G l y c o l i p i d s on T - c e l l

interferon

(19).

I t h a s been o b s e r v e d i n s e v e r a l l a b o r a t o r i e s t h a t i n t e r f e r o n p r o d u c e d i n m i t o g e n - s t i m u l a t e d spleen c e l l s ( T - c e l l interferon) d i f f e r s from f i b r o b l a s t i n t e r f e r o n i n s e v e r a l o f i t s p h y s i c o chemical p r o p e r t i e s , although t h e b i o l o g i c a l e f f e c t s are q u i t e s i m i l a r t o those o f the f i b r o b l a s t v a r i e t y ( 2 0 ) . P r e l i m i n a r y s t u d i e s u s i n g c r u d e i n t e r f e r o n preparations Tappr. 1 0 lU/mg) o b t a i n e d from c u l t u r e d mouse spleen c e l l s o f BCG s e n s i t i z e d a n i m a l s a f t e r s t i m u l a t i o n w i t h o l d t u b e r c u l i n (21) i n d i c a t e d t h a t g a n g l i o s i d e s were much l e s s i n h i b i t o r y t o t h i s i n t e r f e r o n t h a n t o mouse f i b r o b l a s t i n t e r f e r o n ( 2 2 ) . In a n a t t e m p t t o f u r t h e r e l u c i d a t e whether a f f i n i t y f o r g a n g l i o s i d e s i s indeed a p r o p e r t y n o t shared by T - c e l l i n t e r f e r o n , we have c o l l a b o r a t e d w i t h t h e l a b o r a t o r y o f D r . E r n e s t o F a l c o f f and s y s t e m a t i c a l l y compared t h e e f f e c t s o f g l y c o l i p i d s o n a n t i v i r a l and a n t i g r o w t h a c t i v i t i e s o f mouse T - c e l l and f i b r o b l a s t i n t e r f e r o n s u n d e r i d e n t i c a l e x p e r i m e n t a l conditions u s i n g more h i g h l y p u r i f i e d p r e p a r a t i o n s o f t h e f o r m e r (1.6 χ 1 0 lU/mg; r e f . A s seen i n F i g u r e 7, a t g a n g l i o s i d e c o n c e n t r a 3

5

t i o n s where a n t i v i r a l a c t i v i t y o f f i b r o b l a s t i n t e r f e r o n was com p l e t e l y i n h i b i t e d , that o f T - c e l l i n t e r f e r o n remained unchanged. Individual g l y c o l i p i d s that i n h i b i t e d f i b r o b l a s t interferon ( F i g u r e 2) had no e f f e c t when t e s t e d w i t h T - c e l l i n t e r f e r o n u n d e r i d e n t i c a l c o n d i t i o n s a t c o n c e n t r a t i o n s up t o 100 μΜ 0 9 ) . These included G , G , G , G i , G j i b and G . M 3

M 2

M 1

D

a

u

T h a t T - c e l l i n t e r f e r o n does n o t b i n d t o g a n g l i o s i d e s i s d e m o n s t r a t e d by i t s b e h a v i o r o n g a n g l i o s i d e a f f i n i t y c o l u m n s : Under c o n d i t i o n s where o v e r 90% o f mouse f i b r o b l a s t interferon was r e t a i n e d (as shown i n F i g u r e k) T - c e l l i n t e r f e r o n q u a n t i t a t i v e l y e l u t e d i n t h e b r e a k t h r o u g h o f t h e column ( F i g u r e 8). T c e l l i n t e r f e r o n , a f t e r p a s s a g e t h r o u g h t h e a f f i n i t y c o l u m n , was still insensitive to ganglioside i n h i b i t i o n , excluding the p o s s i -


400

C E L L

S U R F A C E


Figure 7. Effects of bovine brain gangliosides on antiviral activities of mouse fibroblast and T-cell interferons Experiment was carried out as described in Figure 1, using 20 IU/mL of both interferons. · , Mouse fibroblast interferon; •, T-cell interferon; M, T-cell interferon after passage through a Sepharose-ganglioside column (see Figure 8). Y : EMC titer in the absence of interferon (19).

0 " 2.5 5 10 20 GANGLIOSIDE CONCENTRATION [AM SIALIC ACID]

Figure 8. Lack of binding of mouse T-cell interferon to Sepharose-ganglioside columns Experiment was carried out at the same time and under the same conditions as described in Figure 4, applying 1 mL of a T-cell interferon solution containing 10 1U. At least 90% of the applied antiviral activity passed the column unretarded (19). 3

2 4 6 ELUTION VOLUME [mL]


22.

A N K E L

E T

A L .

Interferon-

Carbohydrate

Interaction

401

b î l i t y that a non-1nterferon contaminant with high a f f i n i t y f o r g a n g l i o s i d e s had been removed by t h i s p r o c e d u r e (See F i g u r e 7). To e x c l u d e e n z y m a t i c d e s t r u c t i o n o f g a n g l i o s i d e s we i n c u b a t e d a s o l u t i o n o f g a n g l i o s i d e s w i t h T - c e l l i n t e r f e r o n a t an i n t e r f e r o n c o n c e n t r a t i o n 10 t i m e s h i g h e r t h a n t h a t n o r m a l l y used i n t h e a n t i v i r a l a s s a y , f o r 2k h r s a t 3 7 ° , t h e n h e a t - i n a c t i v a t e d t h e T - c e l l i n t e r f e r o n and a s s a y e d t h e t r e a t e d g a n g l i o s i d e s f o r t h e i r i n h i b i t o r y a c t i o n on f i b r o b l a s t i n t e r f e r o n . As c o n t r o l we used a g a n g l i o s i d e s o l u t i o n t r e a t e d i d e n t i c a l l y , but in the absence o f T-cell interferon. A l t h o u g h t h e r e was a s m a l l d e c r e a s e i n i n h i b i t o r y potency o f the g a n g l i o s i d e s o l u t i o n s a f t e r t h i s treatment ( a p p r o x i m a t e l y 30%), t h e r e was no s i g n i f i c a n t d i f f e r e n c e between t h e s o l u t i o n s p r e i n c u b a t e d w i t h T - c e l l i n t e r f e r o n as compared t o t h o s e t h a t were p r e i n c u b a t e d i n MEM a l o n e . Thus i t a p p e a r s t h a t l a c k o f b i n d i n g t o and i n h i b i t i o n by g a n g l i o s i d e s i s due t o t h e T - c e l l i n t e r f e r o n m o l e c u l e i t s e l f and n o t t o c o n t a m i n a t i n g f a c t o r s t h a t e i t h e r compete f o r b i n d i n g t o g a n g l i o s i d e s o r d e g r a d e them t o n o n - i n h i b i t o r y breakdown p r o d u c t s . Effect

of T-cell

Interferon

on Growth o f L - l 2 1 OS and L - l 2 1 OR C e l l s

uw. I f t h e a f f i n i t y o f mouse f i b r o b l a s t i n t e r f e r o n f o r g a n g l i o s i d e s r e l a t e s t o i t s f u n c t i o n a l i n t e r a c t i o n w i t h mouse c e l l s , t h e n c l e a r l y T - c e l l i n t e r f e r o n must i n t e r a c t w i t h d i f f e r e n t comp o n e n t s o f t h e s e c e l l s , a s i t does n o t b i n d t o g a n g l i o s i d e s . T h e r e f o r e , i f t h e L - 1 2 1 0 R c e l l s t h a t were s e l e c t e d f o r t h e i r r e s i s t a n c e t o f i b r o b l a s t i n t e r f e r o n (6), had a l t e r e d o r i n a c c e s s i b l e s i t e s on t h e membrane t h a t no l o n g e r a l l o w e d p r o d u c t i v e i n t e r a c t i o n w i t h f i b r o b l a s t i n t e r f e r o n , then T - c e l l i n t e r f e r o n m i g h t s t i l l be a c t i v e w i t h t h e s e c e l l s . That t h i s i s indeed the c a s e i s shown i n F i g u r e 9. L-121 OS c e l l s were f o u n d t o be e q u a l l y s e n s i t i v e to the a n t i g r o w t h a c t i v i t i e s o f both i n t e r f e r o n s . Howe v e r , L-1210R c e l l s , a l t h o u g h i n s e n s i t i v e to f i b r o b l a s t i n t e r f e r o n , were as s e n s i t i v e t o T - c e l l i n t e r f e r o n as L-121 OS c e l l s . In a d d i t i o n and as e x p e c t e d , a n t i g r o w t h a c t i v i t y o f T - c e l l i n t e r f e r o n , l i k e a n t i v i r a l a c t i v i t y , was found t o be r e s i s t a n t t o i n h i b i t i o n by g a n g l i o s i d e s a t c o n c e n t r a t i o n s t h a t c o m p l e t e l y reversed t h e a n t i g r o w t h e f f e c t o f f i b r o b l a s t i n t e r f e r o n ( F i g u r e 8). That r e s i s t a n c e o f L-121 OR c e l l s t o f i b r o b l a s t i n t e r f e r o n was n o t due to g a n g l i o s i d e s (or o t h e r i n h i b i t o r s s p e c i f i c f o r the f i b r o b l a s t v a r i e t y ) shed f r o m t h e s e c e l l s i n t o t h e medium, was c o n f i r m e d by a s s a y i n g t h e a n t i g r o w t h a c t i v i t y o f f i b r o b l a s t i n t e r f e r o n on L - l 2 1 OS c e l l s s u s p e n d e d i n 4-day o l d c u l t u r e medium o f L - 1 2 1 0 R cells. In c o m p a r i s o n t o c o n t r o l L-121 OS c e l l s s u s p e n d e d i n 4-day o l d c u l t u r e medium from L - l 2 1 OS c e l l s , t h e a n t i g r o w t h e f f e c t o f f i b r o b l a s t i n t e r f e r o n was u n c h a n g e d , i n d i c a t i n g t h a t d i f f e r e n t r e s p o n s e s t o b o t h i n t e r f e r o n s by L-121 OR c e l l s i s due t o t h e c e l l s t h e m s e l v e s and n o t t o f i b r o b l a s t i n t e r f e r o n - s p e c î f i c i n h i b i t o r s shed i n t o t h e medium by L - 1 2 1 0 R c e l l s , i n a c c o r d a n c e w i t h r e s u l t s


C E L L

402

S U R F A C E


Figure 9. Antigrowth activities of mouse fibroblast and T-cell interferons on L-1210S (left) and L1210R cells (right) and the effects of gangliosides on antigrowth activity Cells were seeded at an original density of 8 X 10'' L-l 21 OS and L-l 21 OR cells I mL in 0.3 mL total volume. Cells were cultured as in Figure 5 and counted after three days of growth. The cell number in control cultures (100%) was 3.1 X 10 L-1210S cells/mL and 2.6 X 10 L-1210R cells/mL. O , Mouse fibroblast interferon; · , mouse fibroblast interferon plus gangliosides (52 μΜ sialic acid); •, T-cell interferon; T-cell inter feron plus gangliosides (52 μΜ sialic acid) (19). s

5


22.

A N K E L

E T

previously

A L .

Interferon-

reported

Carbohydrate

403

Interaction

(6).

Discussion Data p r e s e n t e d In t h i s c o m m u n i c a t i o n p r o v i d e t h e f o l l o w i n g evi dence: a) b o t h a n t i v i r a l and a n t i g r o w t h a c t i v i t i e s o f mouse f i b r o b l a s t i n t e r f e r o n a r e i n h i b i t e d by g a n g l i o s i d e s ; b) i n h i b i t i o n i s due t o b i n d i n g o f i n t e r f e r o n t o g a n g l i o s i des ; c) b i n d i n g i n v o l v e s t h e c a r b o h y d r a t e s i d e c h a i n s on t h e g a n g l i o s i d e m o l e c u l e s , and i s a t l e a s t i n p a r t d i r e c t e d t o w a r d s s i a l i c a c i d r e s Îdues ; d) n e i t h e r a n t i v i r a l n o r a n t i g r o w t h a c t i v i t i e s o f T - c e l l i n t e r f e r o n a r e i n h i b i t e d by g a n g l i o s i d e s , and T - c e l l i n t e r f e r o n does n o t b i n d t o g a n g l i o s i d e s ; e) mouse l e u k e m i a L - 1 2 1 0 R c e l l s s e l e c t e d f o r r e s i s t a n c e t o f i b r o b l a s t i n t e r f e r o n r e t a i n unchanged s e n s i t i v i t y t o T - c e l l interferon. It i s n o t known w h e t h e r f i b r o b l a s t o r T - c e l l i n t e r f e r o n s o r p a r t s o f them have t o e n t e r t a r g e t c e l l s i n o r d e r t o r e s u l t i n a n t i v i r a l or antigrowth responses. The f a c t t h a t mouse f i b r o blast interferon interacts with carbohydrate constituents of g a n g l i o s i d e m o l e c u l e s and t h a t some t r a n s f o r m e d mouse c e l l s g a i n i n c r e a s e d s e n s i t i v i t y to i t s a n t i v i r a l e f f e c t a f t e r uptake o f exogenous g a n g l i o s i d e s i n t o t h e c e l l membrane (5) tempts us t o speculate that i n t e r a c t i o n o f t h i s type of i n t e r f e r o n with c e l l membrane g a n g l i o s i d e s i s o f f u n c t i o n a l s i g n i f i c a n c e . Clearly, if t h i s were t h e c a s e , t h e n T - c e l l i n t e r f e r o n , a l t h o u g h p r o d u c i n g t h e same b i o l o g i c a l e f f e c t s as f i b r o b l a s t i n t e r f e r o n , must have a d i f f e r e n t mechanism by w h i c h i t i n t e r a c t s w i t h i t s t a r g e t c e l l s , s i n c e i t does n o t b i n d t o g a n g l i o s i d e s and i s a c t i v e w i t h c e l l s s e l e c t e d f o r r e s i s t a n c e to f i b r o b l a s t i n t e r f e r o n . It i s p o s s i b l e t h a t t h e r e a r e two c l a s s e s o f i n t e r f e r o n b i n d i n g s i t e s on t h e c e l l membrane, e a c h s p e c i f i c f o r p r o d u c t i v e i n t e r a c t i o n w i t h o n l y one t y p e o f i n t e r f e r o n . Thus p r o l o n g e d growth o f L-1210 c e l l s in the p r e s e n c e o f f i b r o b l a s t i n t e r f e r o n c o u l d s e l e c t f o r t h o s e c e l l s t h a t have no o r n o n - f u n c t i o n a l b i n d ing s i t e s f o r f i b r o b l a s t i n t e r f e r o n , but s t i l l c a r r y u n a l t e r e d sites for binding of T - c e l l interferon. A l t e r n a t i v e l y , uptake mechanisms f o r b o t h i n t e r f e r o n s o r t h e i r a c t i v e f r a g m e n t s m i g h t be d i f f e r e n t , one i n v o l v i n g g a n g l i o s i d e s , t h e o t h e r one a d i f f e r e n t t y p e o f g l y c o l i p i d o r none a t a l l . T h i r d l y , a l t h o u g h the b i o l o g i c a l r e s p o n s e s t o b o t h t y p e s o f i n t e r f e r o n a p p e a r t o be i d e n t i c a l , t h e r e m i g h t be two ( o r more) d i f f e r e n t mechanisms by w h i c h t h e s e m i g h t be t r i g g e r e d , i n v o l v i n g a c t i v a t i o n o f d i f f e r e n t enzymes o r e n z y m a t i c s t e p s , e a c h s p e c i f i c f o r one t y p e o f i n t e r feron. A t t h e p r e s e n t t i m e t h e r e i s no d i r e c t e v i d e n c e t o d e c i d e which o f these p o s s i b i l i t i e s is the c o r r e c t o n e . There are

two a s p e c t s

of

medical

significance

related


to

our

a404

CELL SURFACE GLYCOLIPIDS

observations: Firstly it is known that cancer patients ofte have elevated levels of circulating gangliosides which might reflect increased concentrations of these glycolipids in the tumor-surrounding tissue (23,24). Therefore, treatment of such patients with human fibroblast or leucocyte interferon might not be very effective, as these two also bind to gangliosides (5,25). Thus using human T-cell interferon as an alternative treatment in cases where fibroblast or leukocyte interferons fail to show the desired effects could have obvious advantages, provided that indeed the former is comparable to mouse T-cell interferon in its resistance to inhibition by gangliosides. Secondly, the observations of Gresser et_ ah (6) concerning the selection of fibroblast interferon-resistant leukemia cells might be of relevance in interferon therapy of leukemic patients, which likewise might select for resistant cells that would escape from the desired growth inhibition. Our data suggest that alternation between fibroblast and T-cell interferons might be a useful approach to prevent such selection. Acknowledgements This work was supported by grants from the National Science Foundation (PCM 76-84125) and from the National Institutes of Health (AI-15007).

Literature Cited: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Besançon, F . , and H. Ankel, Nature (London) (1974) 250, 784-786. Besançon, F., and H. Ankel, unpublished observation. Besançon, F., and H. Ankel, Nature (London) (1974) 252, 478-480. Besançon, F . , H. Ankel, and S. Basu, Nature (London) (1976) 259, 576-578. Vengris, V . E . , F.H. Reynolds, J r . , M.D. Hollenberg, and P.M. Pitha, Virology (1976) 72, 486-493. Gresser, I., M.T. Bandu, and D. Brouty-Boye, J. Natl. Cancer Inst. (1974) 52, 553-559. Wietzerbin, J., S. Stefanos, M. Lucero, E. Falcoff, D.C. Thang, and M.N. Thang, Biochem. Biophys. Res. Commun. (1978) 85, 480. Wietzerbin, J., S. Stefanos, M. Lucero, Ε. Falcoff, J. O'Malley, and E. Sulkowski, Gen. Virol., in press. Svennerholm, L.J., Neurochem. (1963) 10, 613-623. Folch, J., M.B. Lees, and G.H. Sloane Stanley, J. Biol. Chem. (1957) 226, 497.


22.

11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

ANKEL ET AL.

Interferon- Carbohydrate Interaction

405

Brunngraber, E.G., G. Tettamanti, and B. Berra, In "Glycolipid Methodology" (L.A. Witting, ed.) American Oil Chemists' Society, pp. 159-186 (1976). Sica, V., I. Parikh, E. Nola, G.A. Puca, and P. Cuatrecasas, J. Biol. Chem. (1973) 248, 6543-6558. Cuatrecasas, P., Biochemistry (1973) 12, 4253-4264. Craighead, J . E . and A. Shelokov, Proc. Soc. Exp. Biol. Med. (1961) 108, 823-826. Svennerholm, L . , Biochim. Biophys. Acta (1957) 24, 604-611. Warren, L . , J. Biol. Chem. (1959) 234, 1971-1975. Ledeen, R.W., and R.K. Yu, in "Glycolipid Methodology" (L.A. Witting, ed.) American Oil Chemists' Society, pp. 187-214 (1976). De Maeyer-Guignard, J., M.G. Tovey, I. Gresser, and E. De Maeyer, Nature (London) (1978) 271, 622-625. Besançon, F . , H. Ankel, C. Krishnamurti, and Ε. Falcoff, Manuscript in Preparation (1979). Epstein, L.P. in "Interferons and their Actions", W.E. Stewart II, ed., CRC Press, Inc. pp. 91-132 (1977). Sonnenfeld, G., A.D. Mandel, and T.C. Merrigan, Cellular Immunology (1977) 34, 193-206. Besançon, F . , H. Ankel, G. Sonnenfeld, and C.T. Merrigan, unpublished observation. Kloppel, T.M., T.W. Keenan, M.J. Freeman, and D.J. Morré, Proc. Natl. Acad. Sci. USA, (1977) 74, 3011-3013. Portoukalian, J., G. Zwingelstein, N. Abdul-Malak, and J.F. Doré, Biochem. Biophys. Res. Comm. (1978) 85, 916-920. Besancon, F., and H. Ankel, Texas Rep. on Biol. and Med. (1977) 35, 282-292.

RECEIVED December 10, 1979.


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