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I D E N T I F I C A T I O N O F I N T E R F E R O N - 3 , A S T H E L Y M P H O K I N E
T H A T A C T I V A T E S H U M A N M A C R O P H A G E O X I D A T I V E
M E T A B O L I S M A N D A N T I M I C R O B I A L A C T I V I T Y *
B Y C A R L F . N A T H A N , * H E N R Y W . M U R R A Y , I M I C H A E L E . W l E B E , a n d
B E R I S H Y R U B I N
From The R ockefeller University the Division of lnternational Medicine of the Cornell University
Medica l College and the Lindslev F. Kimball Research Institute of the New Y ork Blood Center
New York 10021; and the Developmental Hematopoiesis Laboratory Sloan-Kettering Institute or
Cancer Research Rye New York 10580
I n t h e l a t e r s ta g e s o f i n f e c t i o n o r a f t e r t h e h o s t r e c o v e r s , l y m p h o c y t e s e n c o u n -
t e r i n g a n ti g e n s o f t h e i n f e c t in g o r g a n i s m c o n f e r u p o n m a c r o p h a g e s a n e n h a n c e d
c a p a c i t y to k il l t h e s a m e o r u n r e l a t e d p a t h o g e n s ( 1) . T h i s p r o c e s s is t e r m e d
m a c r o p h a g e a c t i v a t i o n ( 2 ) . O v e r a d e c a d e a g o , a n t i g e n - s t i m u l a t e d l y m p h o c y t e s
w e r e f o u n d t o r e l e a s e a g l y c o p r o t e i n ( s ) (3 ) t h a t e n h a n c e d t h e a c t i v it y o f t h e
h e x o s e m o n o s p h o s p h a t e s h u n t i n m a c r o p h a g e s ( 4) . S o o n t h e r e a f t e r , s u p e r n a t a n t s
f r o m a n t i g e n - o r m i t o g e n - s t i m u l a t e d l y m p h o c y t e s w e r e s h o w n t o a u g m e n t m a c -
r o p h a g e a n t i m i c r o b i a l a c t iv i ty ( 5 - 9 ). M o r e r e c e n t l y , i t w a s d e m o n s t r a t e d t h a t
l y m p h o i d s u p e r n a t a n t s i n c r e a s e t h e c a pa c i t y o f b o t h m u r i n e ( 1 0 - 1 3 ) a n d h u m a n
( 1 4, 1 5) m a c r o p h a g e s t o s e c r e t e c h e m i c a ll y re a c t iv e , i n c o m p l e t e l y r e d u c e d m e -
t a b o li t e s o f m o l e c u l a r o x y g e n , i n c l ud i n g h y d r o g e n p e r o x i d e . T h e c a p a ci ti e s o f
m a c r o p h a g e s t o s e c r e t e h y d r o g e n p e r o x i d e a n d t o k i l l v a r i o u s m i c r o o r g a n i s m s
a r e c l o s e l y c o r r e l a t e d ( 1 0 , 1 1 ). I n f a c t, r e a c t i v e o x y g e n i n t e r m e d i a t e s a p p e a r t o
m e d i a t e m u c h ( t h o u g h n o t a l l [ 1 5 , 1 6 ] ) o f t h e a n t i m i c r o b i a l f u n c t i o n o f a c t i v a t e d
m a c r o p h a g e s a g a i n st s u c h i n t r a c e l l u la r p a t h o g e n s a s Toxoplasma gondii Trypan o-
soma cruzi Leishm ania Can dida
s p ., a n d m y c o b a c t e r i a ( r e v i e w e d in r e f e r e n c e 1 7) .
T h u s , c e l l - m e d i a t e d i m m u n i t y t o i n tr a c e l l ul a r p a t h o g e n s a p p e a r s t o d e p e n d i n
l a r g e p a r t o n t h e s e c r e t i o n b y l y m p h o c y t e s o f a fa c t or ( s) t h a t a c t iv a t es m a c r o p h a g e
o x i d a t iv e m e t a b o l i s m a n d a n t i m i c r o b i a l a c t i vi ty . I n t h is p a p e r , t h e l y m p h o k i n e
( L K ) 1 m e e t i n g t h is d e s c r i p t i o n is c a l l e d m a c r o p h a g e - a c t i v a t i n g f a c t o r ( M A F ) .
* Supported by grants CA -22090 (to C.F.N.), AI 16963 (to. H.W.M.), and A I 17920 (to B.Y.R.)
from the National Institutes of Health, and grant RF 78021 (to. H.W.M.) from the Rockefeller
Foundation.
*To whom reprint requests should be addressed. Recipient of a research career award from the
Irma T. Hirschl Trust .
lRecipient of a Research Career Development Award in Geographic Medicine from the Rocke-
feller Foundation.
Abbreviations used in this paper: IFN, interferon; IFNa, formerly leukocyte IFN; IFN/3, formerly
fibroblast IFN; IFN 3,, formerly im mune IFN ; LK, lymphokine(s); LPS, bacterial lipopolysaccharide;
MA F, macrophage-activating factor; M LL-IFN 3,, partially purified 1FN3, from human leukocytes
exposed to mez erein and lentil lectin; MN L, peripheral blood mononuclear leukocytes;PMA , phorbol
myristate acetate; R-Hu S, Roswell Park Mem orial Institute m ediu m 1 640 with penicillin, strepto-
mycin, and human seru m ; SEA -IFN'r, partially purified IFN ~, from human leukocytes exposed to
staphylococcal enterotoxin A ; SEM, standard e rro r of the m ean.
67 0 J. Exe. MED. Th e Rockefeller University Press 0022-1 007/83 /09/0670 /20 1.00
Volume 158 September 198 3 670-689
Published September 1, 1983
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NATHAN ET AL 671
T h e s y n th e s is o f M A F b y l y m p h o c y t e s i n tr a c e a m o u n t s , a n d i ts s e c r e t i o n e v e n
b y c lo n e d l y m p h o i d p o p u l a t io n s i n a d m i x t u r e w i th o t h e r L K , h a v e f r u s t ra t e d
a t t e m p t s t o i d e n t i f y i t p h y s i c o c h e m i c a l l y . O n e h y p o t h e s i s is t h a t M A F is in t e r -
f e r o n g a m m a ( IF N 3 ,) . T h e r e is s t r o n g e v i d e n c e t h a t I F N 3 , is t h e L K t h a t i n d u c e s
t h e e x p r e s s i o n o f I a o r D R a n t i g e n s o n M a c r o p h a g e s ( 1 8 , 1 9 ) a n d p r i m e s t h e m
f o r a n t i t u m o r a c t iv i t y ( 2 0 , 2 1) . V a r i o u s I F N s s t i m u l a te m o n o c y t e p l a s m i n o g e n
a c t i v a t o r re l e a s e ( 2 2 , 2 3 ) a n d F c r e c e p t o r e x p r e s s i o n ( 2 4 , 2 5 ). H o w e v e r , n o n e o f
t h e s e e f f e c t s is k n o w n t o b e l i n k e d d i r e c t l y w i th e n h a n c e d a n t i m i c r o b i a l a c t iv i ty .
P a r ti a ll y p u r i f i e d , l e u k o c y t e - d e r i v e d IF N s m a y c o n t a i n o t h e r L K . P o l y d o n a l
a n t i b o d i e s m a y n e u t r a l i z e n o t o n l y I F N b u t t h e c o n t a m i n a n t s a s w e ll . E v e n p r o o f
t h a t c l o n e d , r e c o m b i n a n t I F N c a n a c t i v a t e m a c r o p h a g e s w o u l d n o t e s t a b l i s h t h a t
l y m p h o c y t e s d o s o b y m e a n s o f I F N . F o r a l l t h e s e r e a s o n s , t h e r e l a t i o n s h i p
b e t w e e n M A F a n d I F N h a s r e m a i n e d u n d e f i n e d .
I n th is r e p o r t , w e m a d e u s e o f u n p u r i f i e d a n t i ge n - a n d m i t o g e n - i n d u c e d L K ,
p a r t ia l l y p u r i f i e d l y m p h o c y t e - d e r i v e d IF N 3 ' , p u r e I F N 3 , p r o d u c e d b y b a c t e r i a
c o n t a i n in g t h e c l o n e d h u m a n g e n e ( 26 ), a n d a m o n o c l o n a l a n t i b o d y t h a t n e u -
t ra l iz e s IF N 3 ,. T h i s c o m b i n a t i o n o f r e a g e n t s h a s p e r m i t t e d t h e i d e n t i f ic a t i o n o f
I FN 3 , a s t h e L K t h a t e n h a n c e s b o t h t h e p r o d u c t i o n o f h y d r o g e n p e r o x i d e b y
h u m a n m a c r o p h a g e s a n d t h e i r a b il it y t o k il l a n i n t r a c e l l u la r m i c r o b i a l p a t h o g e n .
M a t e r i a l s a n d M e t h o d s
Culture of Monocvtes. M o n o n u c l e a r l e u k o c y t e s ( M N L ) w e r e i s o la t ed f r o m t h e v e n o u s
b l ood o f norma l adu l t don or s a s desc r i bed (14 , 27). 13-mm d i am g las s cove r s li ps were
pre t r ea t e d fo r 1 wk wi t h 50% HN O3 b e fore t hey we re c l eaned i n e t hano l a s de t a i led (27) .
F or expe r i ment s w i t h H202 r e l ease , 1 X
1 6
M N L s u s p e n d e d i n 0 . 1 m l R P M I - 1 6 4 0
c o n t a i n in g 1 0 0 U / m l p e n i c il li n , 1 00 g / m ] s t r e p t o m y c i n a n d 2 5 % f r e s h - f r o z e n h u m a n
se rum (R-HuS ) were p l a t ed p e r cove r s l i p . Af t e r 2 h , t he cove r s l ips were r i nsed t h r ee t i mes
i n warm E ag l e ' s mi n i mum essen t i a l medi um and t r ans f e r r ed t o 16-mm d i am we l l s i n 24-
we l l t r ays (Cos t a r Da t a P ackag i ng , Cambr i dge , M A) i n 0 .3 ml R-HuS . T he medi um was
rep l aced t he fo l l owi ng day (day 1 ), on day 3 , and eve ry 2 -3 d t he r e a f t e r . F o r expe r i me nt s
wi t h
T. go~tdii
12-mm d i am cover s l i ps r ece i ved 1 .5 x 106 M N L i n R-HuS con t a i n i ng 20%
hea t - i nac t i va t ed human se rum (S ab i n-F e l dman dye t e s t -nega t i ve ) , and were cu l t u r ed i n
groups o f t h r ee cove r s l ips pe r 35-mm d i am p l as t ic pe t r i d ish . Cu l t u r es were r i nsed a f t e r
2 h , a n d g i v e n fr e s h m e d i u m o n d a y 1 a n d e v e r y t h i r d d a y t h e r e a f t e r .
Hydrogen Peroxide Secretiot~.
S e c r e t io n o f H ~ O ~ i n r e s p o n s e to 1 0 0 n g / m l p h o r b o l
myr i s t a t e ace t a t e (P M A) (Conso l i da t ed M i d l ands Co . , B rews t e r , NY) was me asured by t he
f l uorescen t s copo]e ti n a ssay a s desc r i bed (27). T he i ni ti al c onc en t r a t i on o f s copo l e t i n was
se l ec t ed so t ha t
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INTERFERON 7 AS MACROPHAGE ACTIVATING FACTOR
sequen t i a l a f f in i t y chro ma t ogra phy t o a spec i fi c ac t iv i t y o f 1 x 106 an t i v ir a l U / m l ; t hese
a re des i gn a t ed M L L - IF N% Det a il s o f t he pur i f i ca t i on wi l l be fu rn i shed e l sewhere (M .
W i ebe , M . Chang e t a l . , manusc r i p t i n p r epa ra t i on) . Anot he r p r epa ra t i on , t e rmed S E A-
IFN3,, was ob t a i n ed by s t i mul a t i ng buf fy coa t ce l ls w i t h s t aphy l ococca l en t e ro t ox i n A an d
pur i fy i ng t he su pe rn a t an t by sequen t i a l co l um n chr om at og raph y t o a spec if i c ac t i v it y o f
1 x 107 U / m g pro t e i n , a s desc r i bed ( r e f e r enc es 30 , 31 , and B . Y . Rubi n e t a l ., manusc r i p t
i n p r epa ra t i on ) . I n add i t i on , supe rna t an t s w ere co l lec t ed f rom M NL cu l t u r es s t i mul a t ed
for 48 h w i t h concanava l i n A or t oxop l asma l ysa t e a s r epor t ed p r ev i ous l y (15) . Cont ro l
L K w e r e o b t a i n e d b y a d d i n g c o n c a n a v a li n A t o M N L c u l t u r e s a t t h e e n d o f t h e 4 8 - h
i n c u b a t io n o r b y a d d i n g t o x o p l a s m a a n t i g e n a t t h e o u t s e t o f c u l t u r e o f M N L f r o m S a b i n -
F e l dm an dye t e s t -nega t i ve don or s (15). R ecom bi nan t IF N3, syn t hes i zed by
E. coli
(26) was
prov i ded by Gen en t ec h , I nc . , S ou t h S an F ranc i sco , CA. T he l o t used , whi ch gave a s ing l e
band on ana l y t i ca l po l yac ry l am i de ge l e l ec t rophores i s ( pe rsona l comm uni ca t i on , D r . S ang-
He L ee ) , had a spec i f i c ac t i v it y o f 6 X 105 U/ m g. F or cons i s tency , we have a rb i t r a r i l y
expres sed a l l an t i v i ra l t i t e r s in t h i s pape r acc ord i ng t o un i t age o f a s says pe r fo r me d by one
of us (B . R . ) in com par i son t o a l abora t o ry s t anda rd , us i ng a cy t opa t h i c e f f ec t i nh i b i t ion
assay wi th vesicular s tomat i t i s vi rus in WISH (HeLa) cel l s (32) . I t should be noted that
d i f f e r en t an t i v i r a l ac t i v i t i e s were r ecorded fo r some of t hese p r epa ra t i ons i n o t he r
l abora t o r i e s .
Antibodies to IF N .
M onoc l ona l I gGt an t i body GIF -1 neu t r a l i zes human IF N3, bu t no t
IFNc~ or IF NI3 (31) . T h e G IF -1 hyb r i dom a supe rn a t an t used h e re had a neu t r a l i z i ng
a c ti v it y o f 5 , 0 0 0 U / m l . S h e e p g l o b u li n s to h u m a n I F N c ~ a n d t o h u m a n I F N 3 w e r e
N a t i o na l I n s ti tu t e s o f H e a l th ( N I H ) r e f e r e n c e p r e p a r a t io n s G O 2 6 - 5 0 2 - 5 6 8 a n d G O 2 8 -
5 0 1 - 5 6 8 , r e s p e ct iv e l y . T e s t m e d i a w e r e p r e i n c u b a t e d w i th a n t i b o d y f o r 3 0 - 9 0 r a in b e f o r e
use. Pe rce nt inh ibi t ion o f perox ide-re leas in g capaci ty was calculated as 100(1-[PL~+A--PM]
/[PLK--PM]),
w h e r e P = n m o l
H202/mg
c e ll p r o t e i n a f t e r t r e a t m e n t o f m o n o c y t e s a s
i nd i ca t ed by the subsc r i p t s (L K = l ym phok i ne o r IF N3,, A = an t i body , M = m edi um
cont ro l ) .
R e s u L t s
Elimination by Mo~ oclonc~l Anti-IFN3, Antibody of the Abili ty of Unfractionated
Lymphoid Su perna tan t s to Enhance Perox ide Rel ease rom Hu ma n Macrophages .
T h e
1 5 e x p e r i m e n t s s u m m a r i z e d i n T a b l e I a r e i n a c c o r d w i th p r e v i o u s o b s e r v a t io n s
o n t h e c a p a c i t y o f l y m p h o i d s u p e r n a t a n t s g e n e r a t e d w i t h c o n c a n a v a l i n A ( 1 4 ,
1 5 ) o r t o x o p l a s m a a n t i g e n ( 1 5 ) to e n h a n c e t h e p e r o x i d e - r e l e a s i n g c a p a c i t y o f
h u m a n m a c r o p h a g e s . S i m i l a r re s u l ts w e r e s e e n w it h s u p e r n a t a n t s o f b u f f y c o a t
c e ll s p o o l e d f r o m m u l t i p l e b l o o d d o n o r s a n d e x p o s e d t o l e n ti l l e c ti n a n d m e z e r -
e i n. A s e x p e c t e d ( 3 3 - 3 5 ) , t h e s e s u p e r n a t a n t s a ll c o n t a i n e d s u b s t a n t ia l t i te r s o f
I F N ' y ( T a b l e I ). I F N ~ a n d I FN / 3 c o u l d n o t b e d e t e c t e d . T o d e t e r m i n e w h e t h e r
t h e s t i m u l a t o r y a c t iv i ty in t h e s e u n f r a c t i o n a t e d s u p e r n a t a n t s c o u l d b e a s c r i b e d
t o t h e i r c o n t e n t o f I F N y , w e m a d e u s e o f a m o n o c l o n a l a n t i b o d y ( G I F - 1 ) t h a t
n e u t r a l i z e s I F N - y b u t h a s n o d e t e c t a b l e e f f e c t o n I F N0 ~ o r I F N I3 (3 0 ) . I n 8 o f 1 2
e x p e r i m e n t s , G I F - 1 a n t i b o d y r e m o v e d a ll s t i m u l a t o r y a c ti v i ty f r o m t h e s u p e r -
n a t a n ts . I n t h e r e m a i n i n g f o u r e x p e r i m e n t s i t w a s p ar t ia l l y ef f e c ti v e . C o m p l e t e
i n h i b i t i o n w a s s e e n w i th s ix o f t h e s e v e n L K p r e p a r a t i o n s s t u d i e d .
T o r u l e o u t t h e p o s s ib i li ty t h a t G I F - 1 w a s t o x i c, w e a d d e d t h e a n t i b o d y t o
m o n o c y t e s f o r 3 d b e g i n n i n g o n d a y 1 o f c u lt u r e . W h e n t e s t e d o n d a y s 2, 3 , a n d
4 , t h e s e m o n o c y t e s r e l e a s e d 1 0 7 % , 1 1 9 % , a n d 1 1 3 % a s m u c h p e r o x i d e a s t h o s e
c u l t u r e d i n m e d i u m a l o n e ( th e l a t t e r r e l e a s e d 5 2 9 + 1 3 [ d a y 2] , 4 2 0 + 7 [ d a y
3 ], a n d 6 3 0 -+ 7 0 [ d a y 4 ] n m o l / m g c e ll p r o t e i n ) . T h i s d e m o n s t r a t e d t h e l a ck o f
d i r e c t s u p p r e s si v e e f f ec t s o f a n t i b o d y G I F - 1 , a n d f u r t h e r s u g g e s t e d t h a t t h e
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NATHAN ET AL. 67 3
T BLE I
Stimulation of Hum an Macroph age Peroxide-releasing Capacity by Unfractionated Lympho id
Supernatants: Prevention by Mon oclonal Antibody to IFN 'y
Concen- H20~ release by H~O2 release, Inhibition by
Stimuli for LK 1 F N 3 , tration experimental*/ monoclonal
production* titer LK-treated cells*
IFN~, used controla cells antibody
U/m l U/m l nm ol/m g rotein~60
min
Lentil lectin +
mezerein
Prep. 1
100 1-10 321 ,520, 529, 1.5, 1.8, 2.9, 141,311 ~
657, 6964 2.9, 12.0~
Prep. 2 1,000 100 1094 1.7 170
Concanavalin A
Prep. 1 1,000 67-100 339, 1052 2.7, 3.1 85, 89
Pre p. 2 3,000 100-300 368 ,912 2.3, 5.1 49, 104
Prep. 3 1,500 100 511 4.7 105
Control** 0 0 172 ,30 5,3 94 0.8, 0.9, 1.2 ND~
Toxoplasma anti-
gen
Prep. 1 6,000 400 -60 0 514 ,919, 1022 2.3, 4.5, 4.8 78, 113, 146
Prep. 2 2,000 134 529 4.9 95
ControlH 0 0 157, 189 1.2, 1.2 ND
Overall means -4-_
SEM (N): 666 _ 67 (15) 3.8 -+ 0.6 (15) 12 4_ 19 (12)
* MNL from individual donors (for concanavalin A or toxoplasma antigen preparations) or buffy
coat cells pooled from 87 donors (lentil lectin + mezerein) were incubated with the indicated
stimuli and the supernatant s collected as described in Materials and Methods. Prep., preparations
made with different cells on different days. Different batches o f toxoplasma lysate were used for
the two antigen-stimulatedsupernatants.
Human macrophages were exposed to the indicated concentration of LK for 3 d beginning on
the 3rd to 12th d of culture, washed, and stimulated with PMA to measure H202 release.
Cont rol cells were tested in parallel with those in note *, except that LK was not added.
I Antibody GIF-1 used at 60-6 00 U/ml of neutraliz ing capacity for antiviral activity. Inhibition
calculated as described in Materials and Methods.
Values for individual experiments performed on differ ent days or with diff erent donors' macro-
phages, each in triplicate for both H~O2 release and ad herent cell protein. SEM for H20~ release
by triplicates averaged 8.6% of the mean for indiv idual experiments.
** MNL received concanavalin A jus t before harvesting the supernatant, which was added to
macrophages at a 10-fold dilution .
** Not tested.
Supernatan ts from MNL of Sabin-Feldman dye test-negative donors incubated with toxoplasma
antigen were used at a 10% concentra tion on macrophages.
e levated H202-re le as ing capaci ty typ ica lly observed ov er the f i rs t 3 -4 d o f cu l tu re
of adhe ren t MNL may no t be sus ta ined by IFN3, f rom c onta mina t ing T cel ls .
I n t h r ee ex p e r i m en t s , i n cu b a t i o n o f m ac ro p h ag es i n s u p e rn a t an t p l u s an t i -
IFN3, an t ib ody resu l t ed in H202-re leas ing capaci ty substan t i a l ly lower than tha t
o f m ac ro p h ag es i n cu b a t ed i n m ed i u m a l o n e ( i n h i bi t i o n > 1 4 0 % , T ab l e I ). T h i s
sugges ted tha t lymphoid supernatan t s may conta in fac tors suppress ing macro-
phage ox idat ive meta bol i sm. T his possib i li ty was al so ra i sed by the dose-response
prof i l e shown in Fig . 1, in which 30% by vo lume of unfrac t iona ted LK was much
less s t imulatory than 1%.
On the average , adh ere n t ce ll p ro te in was 1 .7 _ 0 .3 t imes grea ter af t er 3 d
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6 7 4
I N T E R F E R O N - y A S M A C R O P H A G E - A C T I V A T I N G F A C T O R
~ o o
1~o0o
E
o
D s o o
O
~ 6
0
i
t~
O
O4 4 11
T
I l l / / /
i 1 1 /
I
I
/
2 0 0
. _
0 3 1 3 1 0 3 0 1 0 0 3 0 0
I F N Y , U l m l
FIGURE 1. En han cem ent of ma crop hage peroxide-releasing capacity by three IFN r-contain-
ing preparat ions and i ts prevention b y monoclonal ant i-IFN~' ant ibody. Hum an m acrophages
were exposed to the indicated concentrat ions of IFN~ on days 4-7 of cul ture, washed, and
st imulated with PMA to measure H20~ release. A n unpu rif ied supernatant fro m buffy coat
cells stimulated with mezerein and lentil lectin (solid triangles) had 102 U /m l antiviral activity.
A fraction obtained from this supernatant (solid circles) had an antiviral activity of 104 U/ml
or t x 10 ~ U /ra g protein. An indep endently purif ied preparat ion from buffy coat cells
stimulated w ith staphylococcal entero toxin A (solid squares) had an activity of 6 x 103 U /m l
or 1 X 107 U /m g protein. T he open symbols are results with the correspondin g preparat ions
exposed to 120- 350 neutralizing U/ m l o f monoclon al ant i-IFN~ antibody. A n equivalent
amo unt of monoclonal ant ibody to a mouse H 2 antigen ( the kind gif t of Dr. R. Steinman, Th e
Rockefeller University) had no effect (not shown). Means _ SEM for triplicates.
i n c u b a t i o n o f m a c r o p h a g e s i n t h e l y m p h o i d s u p e r n a t a n t s t h a n i n c o n t ro l m e d i a
( n = 1 5 ). N e i t h e r t h e e l e v a t i o n s n o r t h e r e d u c t i o n s i n a d h e r e n t c e l l p r o t e i n s e e n
i n i n d i v i d u a l e x p e r i m e n t s w e r e c o n s i s t e n t l y a f f e c t e d b y m o n o c l o n a l a n t i - I F N ' y
a n t i b o d y ( d a t a n o t s h o w n ) .
E f f ec ts o f P ar t i a ll y P ur i f ied IF N y
T h e f o r e g o i n g e x p e r i m e n t s i m p l i e d t h a t
I F N 3 , w a s t h e s o le f a c t o r i n t h e m i t o g e n - o r a n t i g e n - i n d u c e d l y m p h o i d s u p e r n a -
t a n ts t es t e d t h a t c o u l d e n h a n c e h u m a n m a c r o p h a g e p e r o x i d e - r e le a s i n g c a p a c it y .
I f s o , t h e n n a t i v e I F N y s h o u l d h a v e M A F a c t iv i ty . T h e r e s u l ts o f 1 3 e x p e r i m e n t s
w i t h t w o i n d e p e n d e n t l y d e r i v e d p r e p a r a t i o n s e n r i c h e d i n IF N ~ , a r e s u m m a r i z e d
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NATHAN ET AL. 675
i n T a b l e I I . O n t h e a v e r a g e , I F N - r -r ic h f r a c t i o n s e n h a n c e d m a c r o p h a g e p e r o x i d e -
r e l e a s i n g c a p a c i t y 8 . 8 - f o l d . P e r o x i d e r e l e a s e d b y I F N ~ , - t r e a t e d m a c r o p h a g e s o n
d a y s 7 - 9 s o m e t i m e s e x c e e d e d t h a t s e c r e te d b y s a m p le s o f t h e s a m e m o n o c y t e s
o n d a y 0 . M o n o c l o n a l a n t i -I F N ' r a n t i b o d y i n h i b i t e d I F N ~ , - m e d ia t e d e n h a n c e m e n t
o f m a c r o p h a g e p e r o x i d e - r e le a s i n g c a p a c it y b y a n a v e r a g e o f 9 7 % i n s ev e n
e x p e r i m e n t s ( T a b l e I I) . In t w o t i t r a ti o n s o f p r e p a r a t i o n S E A - I F N - r ( Fi g . 1 ), 5 0 %
o f m a x i m a l s t i m u l a ti o n o f m a c r o p h a g e p e r o x i d e - re l e a s in g c a p a ci t y f o ll o w e d
i n c u b a t i o n i n 0 .4 a n d 1 .3 a n t iv i r a l U / m l , a n d p e a k e f f e c t s w e r e s e e n a t 30 a n d
1 0 0
U / m l .
P e r o x i d e - r e l e a s i n g c a p a c i t y u s u a l l y ( b u t n o t i n v a r i a b l y ) p e a k e d o n t h e t h i r d
d a y o f e x p o s u r e t o p a rt i al l y p u r i f i e d I F N % a n d t h e r e a f t e r d e c l i n e d s o m e w h a t ,
d e s p it e r e p l e n i s h m e n t w i t h f re s h I F N % H o w e v e r , p e r o x i d e r e le a s in g - c ap a c i ty o f
c el ls c o n t i n u o u s l y e x p o s e d t o I F N 7 r e m a i n e d m a r k e d l y e l e v a t e d f o r at l e as t 5 d ,
t h e l o n g e s t p e r i o d t e s t e d . F o r e x a m p l e , m a c r o p h a g e s t r e a t e d w i t h I F N ~ , f r o m
d a y s 5 - 8 r e le a s e d 1 , 4 7 9 + 1 2 4 n m o l / m g p r o t e i n / 6 0 m i n , c o m p a r e d t o 3 9 3 _+
2 0 f o r c o n t r o l s . A f t e r 2 m o r e d a y s i n I F N % p e r o x i d e s e c r e t o r y c a p a c i t y fe l l t o
6 5 2 + 5 3 , c o m p a r e d t o 1 0 1 _+ 2 5 f o r t h e c o n t r o l s . H o w e v e r , w h e n I F N 7 w a s
r e m o v e d o n d a y 8 , s e c r e t i o n o n d a y 1 1 fe l l t o 2 3 _+ 4 , c o m p a r e d w i t h 1 2 + 2 i n
t h e c o n t r o l s . T h u s , t h e e n h a n c i n g e f f e c t o f p a r t i a l ly p u r i f i e d I F N ~ , o n m a c r o -
p h a g e p e r o x i d e r e l e a s e w a s r e v e r s ib l e . T h e s e k i n e t i c s c l o se l y p a r a l le l t h o s e
r e p o r t e d e a r l ie r f o r u n f r a c t i o n a t e d l y m p h o i d s u p e r n a t a n t s ( 1 4) .
O n t h e a v e r a g e , c u l t u r e s i n c u b a t e d i n IF N ~ , - ri c h f r a c t i o n s c o n t a i n e d 1 .5 + 0 . 3
t i m e s a s m u c h a d h e r e n t c e ll p r o t e i n a s t h o s e i n m e d i u m a l o n e (n = 1 3 ). T h i s w a s
u n a f f e c t e d b y t h e a n t i b o d y ( n o t s h o w n ).
E f f ec t s o f R e c o m b i n a n t I F N y . T o e s t a b li s h c o n c l u s i v e l y t h a t I F N 3 , c o u l d a c t i v a t e
n i a c r o p h a g e s f o r e n h a n c e d h y d r o g e n p e r o x i d e r e l e a s e , a n d t o e v a l u a t e t h e
p o s si b le c o n t r i b u t i o n o f o t h e r l y m p h o i d c el l p r o d u c t s t h a t m i g h t c o p u r i f y w i t h
TABLE II
S t imula t ion o f H um an Macrophage P erox ide -re leas ing Capac i ty by Par t ia l ly Pur i f ied Na t iv e
IFN y : Preven t ion by Monoc lona l An t ibody to IFN y
H~O~ release by, H202 release
Specific ac- C on ce n- IFN-treated cells
Preparat ion t iv i ty U/ t ra t ion (nmol/mg p r o - exper imental*
mg protein) IFN~,u se d tein/6 0 min) control cells
Inhibition
by mono-
clonalanti-
body
ML L-IFN',/** 1 X 106
SEA-IFN3 ~ 1 X
1 7
Overall means +
SEM (n):
U / m l
10-242
30-300
%
392 ,482,7 06, 2.0, 6.8, 7.1, 12.7~ 10 2, 1241
12261
211 ,367,3 81, 2.0, 3.5, 5.3, 6.5, 41, 80, 103,
652, 1168, 8.9, 11.4, 15.1, 108, 122
1236, 1479, 24.5, 435
1549, 2102
919 _ 155 (13) 8.8 _ 1.8 (12)M 97 _+ 10 (7)
,0ll See Table I. SEM for H202 release by triplicate cultures averaged 9.5% of the mean in individual
experiments.
LK induced w ith mezerein + lentil lectin and fractionated as described in Materialsand Methods.
** LK induced wi th staphylococcalenterotoxin A and fractionated as described in M aterials and
Methods.
Excludes the highest va lue (435).
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I N T E R FE R O N - ') , A S M A C R O P H A G E - A C T I V A T I N G F A C T O R
I F N y , w e n e x t t e s te d t h e e f f e ct s o f p u r e I F N y p r o d u c e d b y b a ct e r ia t r a n s f o r m e d
w i t h th e c l o n e d b u m a n g e n e f o r t h is L K . T h e p o t e n t M A F a c t iv i ty o f r e c o m b i n a n t
IF N ' y i s i l l u s t r a t e d fo r t h r e e i n d e p e n d e n t t i t r a t i o n s i n F i g . 2 . T h e p o s i t i o n s o f
t h e t h r e e d o s e - r e s p o n s e c u rv e s v a r i e d c o n s i d e ra b l y . Ha l f -m a x i m a l s t i m u l a t i o n o f
m a c r o p h a g e p e r o x i d e - r e l e a s i n g c a p a c it y fo l l ow e d 3 d o f e x p o s u r e t o 0 . 0 0 9 , 0 . 14 ,
a n d 1 .3 U / m l o f I F N T , w i t h p e a k r es p o n se s a t 0. 3 , 1 0 , a n d 1 0 0 0 U / m l . T h e
v a r i a b il i ty i n t h e d o s e - r e s p o n s e c u rv e s m i g h t b e d u e i n p a r t t o t h e u s e o f d i f f e r e n t
d o n o r s f o r t h e m a c r o p h a g e s a n d t h e s e r u m i n e a c h e x p e r i m e n t . A n o t h e r p o ss ib le
c o n t r i b u t o r y f a c t o r i s t h e f a c t t h a t t h e t i m e t o p e a k r e s p o n s e v a r i e d f r o m 2 d o f
e x p o s u re (n o t s h o wn ) t o a s l o n g a s 4 d (F i g . 3 ) . W i t h c o n t i n u o u s e x p o s u re t o
IF N p e ro x i d e - r e l e a s i n g c a p a c i t y r e m a i n e d e l e v a t e d fo r a t le a s t 6 d , b u t f e ll
t o w a rd b a s e l i n e w i t h i n 3 d o f r e m o v i n g t h e r e c o m b i n a n t IF N ' r (F ig . 3 ), a s a l r e a d y
n o t e d f o r n a ti v e I F N I n t h e e x p e r i m e n t il l u st r a te d in F ig . 3 , s o m e o f t h e
m a c r o p h a g e s w e r e e x p o s e d to 1 00 U / m l o f I F N 7 f o r 1 0 m i n o n d a y 6 o f c u l t u r e ,
e x t e n si v e l y w a s h e d t o r e m o v e I F N y , a n d t h e n c u l t u r e d i n s t a n d a r d m e d i u m f o r
3 m o re d a y s . T h e i r p e ro x i d e - r e l e a s i n g c a p a c i t y wa s a t l e a s t a s g r e a t a s t h a t o f
0
1
2
oL< J
- 3 - i -; o i i 3
I F N ~ I ~ l o U / m l )
F IG U RE 2. E n h a n c e m e n t o f m a c r o p h a g e p e r o x i d e - r e l e a s i n g c a p a c i t y b y p u r e , r e c o m b i n a n t
1 F N 3, . T w o e x p e r i m e n t s a r e s h o w n i n w h i c h m a c r o p h a g e s w e r e e x p o s e d t o t h e i n d i c a t e d
c o n c e n t r a t i o n s o f I F N y o n d a y s 6 - 9 ( o p e n c i rc l e s a n d t r ia n g l es ) a n d o n e e x p e r i m e n t i n w h i c h
e x p o s u r e w a s f r o m d a y s 5 - 8 o f c u l t u r e ( o p e n s q u a r e s ). T h e d a s h e d l i n e ( so l id tr i a n g l e s )
i n d i c a t e s r e s u l t s in t h e p r e s e n c e o f m o n o c l o n a l a n t i b o d y t o n a t i v e I F N y a d d e d a t ( f r o m l e f t t o
r i g h t ) 3 , 1 0 , o r I 0 0 t i m e s t h e n e u t r a l i z i n g a c ti v i t y f o r t h e i n d i c a t e d c o n c e n t r a t i o n o f n a t i v e
] F N 7 . M e a n s o f t r i p l ic a t e s a r e i n d i c a t e d . S E M s a v e r a g e d 1 3 % o f t h e m e a n .
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N A T H A N E T A L.
6 7 7
0oo o
C
L 6 0 0
Q
0 1
/ I l l \ \
2 0 o e
6 7 8 9 10 11 12
ays
F IG U R E 3 . K i n e t i c s o f r e s p o n s e to r e c o m b i n a n t 1 F N T . M a c r o p h a g e s o n d a y 6 o f c u l t u r e w e r e
i n c u b a t e d i n s t a n d a r d m e d i u m s o li d c i rc l es ) o r 1 0 0 U / m l o f I F N 7 o p e n c ir c le s ) f o r t h e
i n d i c a t e d t im e s b e f o r e a s s ay o f H 2 0 ~ r e l e a se . F r e s h m e d i u m o f t h e s a m e t y p e w a s a d d e d o n
d a y 8 o r 9 f o r c e l ls as s a y e d o n d a y s 1 1 o r 1 2 , re s p e c t i v e l y. T h e d a s h e d l i n e i n d i c a t e s r e s u l t s
f o r c el ls w h i ch w e r e r e m o v e d f r o m I F N 7 o n d a y 9 , w a s h e d f o u r t i m e s i n s t a n d a r d m e d i u m ,
t h e n i n c u b a t e d i n t h e l a t t e r f o r 3 d b e f o r e a s s a y. T h e o p e n t r i a n g l e i n d i c a t e s c e l ls w h i c h w e r e
p u l s e d w i th 1 0 0 U / m l I F N 7 f o r 1 0 m i n o n d a y 6 , w a s h e d f o u r t im e s , a n d i n c u b a t e d in
s t a n d a r d m e d i u m u n t i l a s s a y o n d a y 9 . M e a n s + S E M o f tr i p l ic a t e s .
c e ll s e x p o s e d c o n t i n u o u s l y t o 1 0 0 U / m l I F N ~, f o r t h e e n t i r e 3 - d p e r i o d . R e s u l t s
w e r e s i m i l a r i n a s e c o n d e x p e r i m e n t u s i n g a 2 - h p u l s e .
I n c o n t r a s t t o t h e r e s u l t s w i t h n a t i v e I F N T , m o n o c l o n a l a n t i b o d y G I F - 1 h a d
n o i n h i b i to r y e f f e c t o n t h e M A F a c t i vi ty o f r e c o m b i n a n t I F N T . T h i s is i l lu s t ra t e d
i n Fi g. 2 w i th a n t i b o d y c o n c e n t r a t i o n s r a n g i n g f r o m 3 to 1 0 0 ti m e s t h e a m o u n t
r e q u i r e d t o n e u t r a l i z e a n e q u i v a l e n t a n t iv i r a l a c ti v i ty o f n a t i v e I F N T . A n t i b o d y
G I F - 1 a l s o f a il e d to i n h i b it t h e a n t i v i ra l a c t i v i ty o f r e c o m b i n a n t I F N 3 , d a t a n o t
s h o w n ) .
T h e r e m a r k a b l e p o t e n c y o f r e c o m b i n a n t I F N 7 c o m p a r e d w i t h, n at iv e I F N 7 i n
s o m e e x p e r i m e n t s r a i s e d t h e p o s s ib i l it y t h a t t h e f o r m e r m i g h t c o n t a i n a c o -
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6 7 8
IN T ER F E R O N -3 , A S M A C R O P H A G E -A C T IV A T IN G F A C T O R
s t i m u l a t o r o r t h e l a t t e r m i g h t c o n t a i n a s u p p r e s s o r o M A F a c t i v it y . T o t e s t th e s e
p o s si b il it ie s , m i x i n g e x p e r i m e n t s w e r e c a r r i e d o u t a s s h o w n i n T a b l e I I I . A n
e f f e c ti v e b u t s u b o p t i m a l d o s e o f r e c o m b i n a n t I F N 3, d i d n o t g i v e g r e a t e r t h a n
a d d i t i v e e f f e c t s w h e n c o m b i n e d w i t h a s u b o p t i m a l b u t e f f e c t i v e d o s e o f t h e n a t i v e
p r o d u c t . A n o p t i m a l d o s e o f n a t i v e I F N 3 ,, w h e n n e u t r a l i z e d b y m o n o c l o n a l
a n t i b o d y a n d a d d e d t o r e c o m b i n a n t I F N 3 ,, d id n o t g i v e l es s t h a n a d d i t i v e e f f e c t s.
T h u s , n e i t h e r c o s t i m u l a t o r n o r s u p p r e s s o r f a c t o rs c o u ld b e d e t e c t e d b y m i xi n g .
S e c r e t io n o f h y d r o g e n p e r o x i d e b y m a c r o p h a g e s w a s u n d e t e c t a b l e o r b a r e ly
d e t e c t a b l e a f t e r i n c u b a t i o n i n e i t h e r n a t i v e o r r e c o m b i n a n t I F N 3 , , u n l e s s a
s e c r e t a g o g u e s u c h as P M A w a s a d d e d ( d a ta n o t s h o w n ).
h T d u c t i o n o f A n t i t o x o p l a s m a A c ti vi ty ,.
T h e a b o v e e x p e r i m e n t s e s t a b li s h e d t h a t
I F N 3, e n h a n c e s t h e o x i d a ti v e m e t a b o l i s m o f h u m a n m a c r o p h a g e s . W e n e x t t e s t e d
w h e t h e r I F N ~ a l s o a u g m e n t s t h e a n t i m i c r o b i a l ac t iv i ty o f m a c r o p h a g e s a g a i n st
a n i n tr a c e l lu l a r p a t h o g e n - - t h e c a r d i na l c r i te r i o n f o r m a c r o p h a g e a c t i v at i on . A s
a t e s t o r g a n i s m , w e u s e d t h e p r o t o z o a n , T . g o n d i i . T h e k i ll i ng o f t hi s p a r a s i t e b y
m u r i n e m a c r o p h a g e s i s c l o se l y r e l a t e d t o t h e i r c a p a c i t y to s e c r e t e h y d r o g e n
p e r o x i d e (1 1 ) a n d is m e d i a t e d l a r g e l y b y o x i d a t i v e m e c h a n i s m s ( 1 1 , 2 9 ), b u t t o
s o m e e x t e n t b y o x y g e n - i n d e p e n d e n t p r o c e s s e s as w e l l ( 1 6, 3 6 ).
A s s h o w n i n F ig . 4 , u n s t i m u l a t e d h u m a n m a c r o p h a g e s c u l t u r e d f o r 1 3 - 2 3 d
b e f o r e i n f e c t i o n k i l le d > 5 % o f i n g e s t e d t o x o p l a s m a s i n th e f i rs t 4 h , a n d
s u p p o r t e d i n t r a c e l l u l a r r e p l i c a t i o n ( 5. 5 t o x o p l a s m a s / v a c u o l e a t 2 0 h) . I n c o n t ra s t ,
w h e n t h e c e ll s w e r e p r e i n c u b a t e d f o r 3 d w i t h u n f r a c t i o n a t e d m i t o g e n - o r
TABLE I l l
Evide~,ce Ag ains t a Suppressor Factor in Partia lly Purif ied I FN 3, or a Costimulator
in R ecombina l~ t IF N 3 *
Predicted value
H~O2, nm ol/m g for additive ef-
SEA-IFN3,* Recom. Aby| pr ote in/ 60 min fects~
U / m l U / m l U / m l
0 0 0 15_+ 11 *
6 0 0 178 + 16
30 0 0 367 _+ 16
30 0 100 85 _+ 8
0 0.1 0 139 _+ 28
0 0.3 0 313 _
0 0.3 3 313_+ 11
0 1.0 0 332 + 8
0 1.0 100 320 _+ 9
6 0.1 0 199 _+ 13
30 0.3 100 448 _+ 12
30 1.0 100 478 _+ 8
302
383
390
* Macrophages were treated with IFN3, from days 6-9 of culture.
* Partially purified, na tive IFN 'y elicited with staphylococcal enterotoxin A (see Ta ble
II).
Recom binant IFN3,.
I Mon oclonal antibody G |F- I (does not neutralize recombinant IFN3,).
([SEA-IFN3 + A by ]'- 15) + (Rec om. - 15) + 15, where 15 is the value in medium
alone.
** Me ans --- SEM for triplicates.
Published September 1, 1983
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10/20
LK
n o n e
Co n
A
II
II
II
t o x o
II
II
S E A
Re co m.
A b y
a n t i - T
a n t i - T
a n t i - ~
ant i - /3
a n t i - T
an t i -~x
a n t i - / 3
a n t i - f
No
NATHAN ET AL. 679
P e r c e n t t o x o p l a s m a s k ille d a t 4 h
1
2 3 4 5 6
exp
7
1 -
2 I -
4
I l I -
I I I I l l l l l l l l l I l l l I l l l l l l l l l l l l l I I
1
1 t
2 _ . L L 1 2 L L 2 2 2 . 2 . . . . . . L i . .. .. .. .. H ~II~ L
2 .. .. . IL.,..._
I
4
~ "
7
FIC;URE 4. Toxop lasmacidal activity of macroph ages incubated in lympho kine (LK) prepa-
rations with or w ithout antibody to the indicated types of IFN . Incubations we re in 100-300
U/m l IFNy for 2-3 d beginning on days 13-2 3 of culture. M onoclonal anti-IFN3, antibody
was used at 300-600 neutralizing U /ml. Anti-IFNa and anti-IFN# antibodies were used at
600 neutralizing U/m l. Unpurif ied LK w ere induced with concanavalin A
Con A)
or toxo-
plasma antigen
toxo).
Partially purified n ative IFN3~preparation SEA-IFN3 had an activityo f
l0 T U/ mg protein.
Recom. ,
recombinan t IFN3'. Mean s + SEM for the indicated number of
experiments.
a n t i g e n - i n d u c e d L K o r p a r t ia l ly p u r i f ie d n a t i ve I F N % t h e y k i ll e d 3 8 - 6 0 % o f th e
i n i ti a ll y i n g e s t e d p a r a s i te s w i t h i n 4 h , a n d i n h i b i t e d t h e r e p l i c a t i o n o f s u rv i v i n g
o r g a n i s m s (1 . 8 - 1 . 9 p a r a s i t e s / v a c u o l e a t 2 0 h ) . T h e i n d u c t i o n o f a n t i p a r a s i ti c
a c t iv i ty w as a b r o g a t e d b y t r e a t m e n t o f th e u n f r a c t i o n a t e d s u p e r n a t a n t s o r t h e
p a r t i a l l y p u r i f i e d I F N 3 ' w i t h m o n o c l o n a l a n t i - I F N 3 , a n t i b o d y , b u t n o t w i t h
p o l y c l o n a l s h e e p a n t i b o d i e s a g a i n s t h u m a n I F N ~ o r I F N /3 (F i g . 4 ). D i r e c t e x p o -
s u r e t o 1 0 % c o n c a n a v a l i n A - i n d u c e d L K o r t o 3 0 0 U / m l o f p a r ti a ll y p u r i f i e d
n a t i v e I F N y f o r t h a t 3 7 C d i d n o t a f f e c t t h e a b i l i ty o f t o x o p l a s m a s t o s u r v i v e
a n d r e p l i c a t e w h e n s u b s e q u e n t l y i n g e s t e d b y u n s t i m u l a t e d m a c r o p h a g e s ( d a t a
n o t s h o w n ) .
P u r e , r e c o m b i n a n t I F N 3 , a ls o m a r k e d l y e n h a n c e d m a c r o p h a g e a n t i t o x o p l a s m a
a c t i v it y ( F ig . 4 ) i n a d o s e - d e p e n d e n t m a n n e r ( F i g . 5 ). I n t h e e x p e r i m e n t i l l u s t r a te d
i n F i g . 5 , m a c r o p h a g e s t h a t h a d b e e n i n c u l t u r e f o r 1 3 d w e r e e x p o s e d t o I F N 3 ,
f o r 3 m o r e d a y s b e f o r e c h a l l e n g e w i t h t h e p a r a s it e . U n d e r t h e s e c o n d i t i o n s ,
t o x o p l a s m a c i d a l a c t i v i t y w a s e v i d e n t a f t e r t r e a t m e n t w i t h a s l i t t l e a s 1 U / m l
I FN 3 ,. A d d i t i o n o f 3 00 U / m l o f r e c o m b i n a n t I F N 3 ' h a d n o e f f e c t w h e n i t w a s
a d d e d a f t e r i n f e c ti o n o f t h e m a c r o p h a g e s ( n o t sh o w n ) .
A s w i th e n h a n c e m e n t o f p e r o x i d e - r e l e a s i n g c a p a c i t y , t h e r e w a s v a r i a b il i ty in
t il e o p t i m a l p e r i o d o f p r e in c u b a t i o n i n r e c o m b i n a n t I F N y t o a c t iv a t e m a c r o p h a g e
Published September 1, 1983
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7/18/2019 J Exp Med-1983-Nathan-670-89
11/20
6 8 0
I N T E R F E R O N - y A S M A C R O P H A G E - A C T I V A T I N G F A C T O R
l -
0
5 0
4 0
O~
E o
0
~ o
e-
u
L. 10
0 0.1 1 10 1
6
I
o
Ul
. 4
13.
O ' 0
1 2 3
Da ys incub a t ion in IFN~
F IG U R E 6 . T i m e c o u r s e o f i n d u c t i o n o f t o x o p l a s m a c i d a l ( s o li d c i r c le s ) a n d t o x o p l a s m a s t a t i c
( o p e n t r ia n g l es ) a ct i vi ty o f m a c r o p h a g e s e x p o s e d t o 3 0 0 U / m [ r e c o m b i n a n t I F N y b e g i n n i n g
o n d a y s 1 0 , 1 6 , o r 2 3 o f c u l t u r e . M e a n s + - S E M f o r t h r e e e x p e r i m e n t s a t e a c h t i m e p o i n t
e x c e p t t w o e x p e r i m e n t s a t 3 d .
l o gi c a lt e r a ti o n s d i d n o t a p p e a r t o b e n e c e s s a r y f o r e n h a n c e d p e r o x i d e - r e l e a si n g
capac i ty .
D i s c u s s i o n
W e c o n c l u d e t h a t I F N 7 is a p o t e n t a c t i v at o r o f h u m a n m a c r o p h a g e o x id a t iv e
m e t a b o l i s m a n d a n t i m i c ro b i a l a c t iv i t y . U n d e r t h e c o n d i t i o n s t e s t e d , t h e o n l y s u c h
a c t i v a to r c o n s is t e n tl y d e t e c t e d i n t h e m e d i u m o f a n t i g e n - o r m i t o g e n - s t i m u l a t e d
h u m a n l e u k o c y t e s w a s IF N 3 '. I F N 7 is t h e f i rs t s e c r e t o r y p r o d u c t o f T i y m p h o c y t e s
(3 3 , 3 4 , 3 9 , 4 0 ) o f k n o w n s t ru c t u re (4 1 , 4 2 ) . T h u s i t i s o f s p ec i a l i n t e r e s t t h a t i t
c a p a c i t a t e s m a c ro p h a g e s t o r e l e a s e a m i c ro b i c i d a l p ro d u c t (H2 0 2 ) wh o s e c h e m i c a l
c o m p o s i t i o n i s a l so k n o w n .
IFN-) , i s the LK fo r wh ich the mos t sens i t ive b ioassay i s ava i lab le , namely ,
i n d u c t i o n o f a n t iv i r al r e si s ta n c e . W i t h r e c o m b i n a n t I F N a c t iv a t io n o f m a c r o -
p h a g e p e r o x i d e - r e l e a s i n g c a p a c i ty w a s s t i m u l a t e d t o 5 0 o f th e m a x i m a l v a l ue
w i t h a g e o m e t r i c m e a n c o n c e n t r a t i o n o f 0 .1 a n t i v ir a l U / m l . A s s u m i n g t h a t
r e c o m b i n a n t I F N 7 is a n o n g l y c o s y l a t e d d i m e r o f 3 4 , 2 9 2 d a l to n s , t h e n t h e 5 0
m a x i m a l l y e f f e c t iv e c o n c e n t r a t i o n o f t h e p r e p a r a t i o n u s e d h e r e c a n b e e s t i m a t e d
a t 0 . 4 - 6 3 p i c o m o l a r ( g e o m e t r i c m e a n , 6 p M ) . T h e r e m a r k a b l e s e n s it iv i ty o f t h e
o x i d a t iv e m e t a b o l i s m o f h u m a n m a c r o p h a g e s t o e n h a n c e m e n t b y I FN 3 , s u g g es t s
t h a t m a c r o p h a g e a c t iv a t io n m a y b e o n e o f t h e p r i m a r y p h y s io l o gi c f u n c ti o n s o f
th i s LK.
I t is n o w u n a r g u a b l e t h a t m o r e t h a n o n e L K a c t i vi ty (a s d e f i n e d b y e f f e c t s o n
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682 INTERFERON--y AS MACROPHAGE-ACTIVAT1NG FACTOR
FIGURE 7. Morphology of IFNT-treated macrophages by phase contrast microscopy. A
control cells in standard medium on day 8. B macrophages exposed from days 5-8 to partially
puri fied native IFNT. C same as B but a4so exposed to monoclonal antibody against IFN3 . D
treated from days 6-12 with 100 U/ml recombinant IFN3 .
differ ent targe t cells) can be ascribed to the same molecule. Thus, IFN3, induces
antiviral activity in a variety of cell types and enhances the capacity of macro-
phages to secrete H202 and to kill toxoplasmas. In addition, it is now clear that
a single LK can cause pleiotropic effects in the same cell population. For example,
the recom binan t IFN3, used in our studies has also been re port ed to enh ance the
expression of DR antigens (19) and Fc receptors (25) on monocytes, and to
activate monocytes to kill tumor cells (43).
Four qualifications require emphasis. First, the term MAF has been applied
to factors that induce a variety of physiologic changes in macrophages. We have
used the term in a restricted sense, and our results do not bear on the issue
whether MAF as defined in other ways or in other species is IFN7 (reviewed in
refere nce 21). Second, our studies are fully compatible with the possibility that
ther e may be o ther factors besides IFN3, that can in duc e the same changes in
macrophages as described here. IFNa, a product of non-T leukocytes, is of
special interest in this regard (C. Nathan, H. Murray, B. Rubin, and M. Wiebe,
unpublished observations). Our experiments do suggest that other MAFs were
either not consistently present in active amounts in the lymphoid supernatants
we studied, or if present, were masked by inhibitors or depen den t on IFN3, in
order to activate macrophages. Third, as already noted, the biochemical and
functional changes we measur ed in macrophages in response to IFN3, are only a
few of an unknown number of alterations induced in the same cells by the same
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N A T H A N E T A L 6 8 3
m o l e c u l e . E x p e r i m e n t s a r e i n p r o g r e s s t o d e t e r m i n e w h e t h e r t h e l y m p h o k i n e
t h a t e n h a n c e s o x y g e n - i n d e p e n d e n t a n t i m i c r o b ia l a c ti v it y i n h u m a n m a c r o p h a g e s
(1 5 , 1 6) m a y a l s o b e IF N % F i n a l ly , we h a v e n o t fo rm a l l y e x c l u d e d t h e p o s s ib i li ty
t h a t t h e e f f e c t s o f I F N 3, o n m a c r o p h a g e s c o u l d h a v e b e e n m e d i a t e d b y a n o t h e r
t y p e o f c e ll c o n t a m i n a t i n g t h e c u l t u re s . H o w e v e r , r e s u lt s w e r e s i m i l a r w h e t h e r
I F N ~, w as a d d e d s t a r t i n g o n t h e 3 r d t h r o u g h t h e 2 3 r d d a y o f c u l t u r e . I n m o s t
o f t h e s e c u l tu r e s , n o c e l ls o t h e r t h a n m a c r o p h a g e s c o u l d b e i d e n t if i e d b y
m o r p h o l o g i c c r i te r i a.
N e u t r a l i z a t io n o f M A F a c ti v it y in c r u d e l y m p h o i d s u p e r n a t a n t s b y t h e m o n o -
c l o na l an t i- I FN ~ , a n t i b o d y w a s s t r o n g e v i d e n c e t h a t M A F w a s I F N % H o w e v e r ,
t h e e p i t o p e s r e c o g n i z e d b y m o n o c l o n a l a n t ib o d i e s m a y s o m e t i m e s b e s h a r e d b y
s e e m i n g l y u n r e l a t e d p r o t e i n s ( 4 4) . A c c o r d i n g l y , i t w as n e c e s s a ry t o d e t e r m i n e
d i r e c t ly w h e t h e r n a t i v e, l e u k o c y t e - d e r i v e d I F N ~ h a d M A F a c t iv i ty . U s i n g t w o
i n d e p e n d e n t p r o to c o l s f o r i n d u c t i o n a n d p a r ti a l p u r if i c a ti o n o f I F N % M A F
a c ti v it y w as r e a d i ly d e m o n s t r a t e d , w a s e n r i c h e d t o a t l e a s t th e s a m e d e g r e e a s
a n t i v i r a l a c t i v i t y , a n d wa s a g a i n n e u t r a l i z e d b y m o n o c l o n a l a n t i - IF N~ , a n t i b o d y .
No n e t h e l e s s , t h e s e p r e p a ra t i o n s o f IF N r we re n o t p u re , a n d t h e p o s si b il it y
r e m a i n e d t h a t a c o n t a m i n a n t c o n t r i b u t e d t o M A F a c t i v i t y . F o r a d e f i n i t i v e
a n s w e r , w e t u r n e d t o a p r e p a r a t i o n i n w h i c h IF N ~ , w a s t h e o n l y h u m a n p r o t e i n
a n d p r o b a b l y t h e o n l y p r o t e in , n a m e l y , IF N ~ , p u r i f i e d t o a p p a r e n t h o m o g e n e i t y
f r o m b a c t e r i a t r a n s f o r m e d w i th t h e c l o n e d g e n e f o r h u m a n I FN 3 ,. T h i s p r e p a -
r a t i o n d i s p la y e d e s pe c ia l ly p o t e n t M A F a c t i v it y . T h e t i m e r e q u i r e d f o r m a c r o ,
p h a g e s t o d i sp l ay a n o p t i m a l r e s p o n s e t o r e c o m b i n a n t I F N ~, v a r ie d f r o m 2 - 4 d .
E x p o s u re s t o IF N~ ' a s b r i e f a s 1 0 -1 2 0 m i n l e d t o s u b s ta n t i a l a c t i v a t i o n wh e n
m a c r o p h a g e s w e r e t e s t e d 3 d la t e r. A f t e r 3 d o f e x p o s u r e t o I F N % p e r o x i d e
s e c re t o ry c a p a c i t y f e l l t o b a re l y d e t e c t a b l e l e v e l s wh e n IF N~ , wa s r e m o v e d , b u t
wa s e l e v a t e d fo r a t l e a s t 6 d wh e n IF N~ , r e m a i n e d .
M o n o c l o n a l a n t i b o d y G I F - 1 n e u t r a l i z e d b o t h t h e a n t iv i ra l a n d m a c r o p h a g e -
a c t i v a t i n g e f f e c t s o f n a t i v e IF N~ ,, y e t n e u t r a l i z e d n e i t h e r o f t h e s e e f f e c t s o f t h e
r e c o m b i n a n t b a c t e r ia l p r o d u c t . T h e r e a r e b e l i e v e d t o b e tw o c a r b o h y d r a t e c h a in s
o n n a t i v e IF N 7 (2 6 , 4 1 , 4 2 ) a n d n o n e o n t h e r e c o m b i n a n t p ro d u c t . I t is p o s s ib l e
t h a t t h e c a r b o h y d r a t e g r o u p s c o n t r i b u t e t o t h e e p i t o p e s e e n b y t h e a n t i b o d y .
T h e s e c a r b o h y d r a t e s a r e n o t n e c e s s a r y f o r t h e e x p r e s s i o n o f a t l e as t s o m e o f th e
a n t iv i r al ( 4 5) o r M A F a c t iv i ty o f I F N % H o w e v e r , t h e c a r b o h y d r a t e m o i e t ie s m a y
b o rd e r a d o m a i n n e c e s s a ry fo r IF N~ , a c t i v i t y , s u c h a s a r e c e p t o r -b i n d i n g s i t e .
T h e a n t i b o d y m i g h t i n t e r f e r e w i t h t h e f u n c t i o n o f t h is d o m a i n b y s te r i c hi n -
d r a n c e .
R e c o m b i n a n t I F N ~, w a s us u a ll y c o n si d e r a b ly m o r e p o t e n t t h a n t h e e q u i v a l e n t
c o n c e n t r a t i o n o f n a t i v e IF N ' y , b a s e d o n a n t iv i r a l a c ti v it y . T h e r e a r e a t l e as t t h r e e
p o s s i b l e e x p l a n a t i o n s fo r t h i s a p p a re n t d i s c r e p a n c y . F i r s t , t h e p a r t i a l l y p u r i f i e d
n a t i ve I F N ~ ' c o u l d h a v e b e e n c o n t a m i n a t e d w i t h a f a c t o r s u p p r e s si n g m a c r o p h a g e
o x i d a t i v e m e t a b o l i s m . S u c h a su p p re s s i v e f a c t o r h a s b e e n o b s e rv e d i n t h e c u l t u r e
m e d i u m o f m u r i n e l y m p h o i d c e ll s ( 4 6) , a n d i n h i b i t o r s o f IF N a n t i v i r a l a c t i v it y
a r e r e p o r t e d l y p r o d u c e d b y s t i m u l a te d l y m p h o c y t e s ( 47 ). T h e a b il it y o f m o n o -
c l o n a l a n t i b o d y GIF -1 t o n e u t r a l i z e n a t i v e IF N~ , w i t h o u t n e u t r a l i z i n g r e c o m b i -
n a n t IF N ' y m a d e i t p o s s i b l e t o p e r fo rm m i x i n g e x p e r i m e n t s , i n wh i c h n a t i v e
I F N 3, p r e p a r a t i o n s p l us a n t i b o d y G I F - 1 w e r e a d d e d t o r e c o m b i n a n t I F N % N o
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6 8 4 INTERFERON- r AS MACROPHAGE-ACTIVATING FACTOR
s u p p re s s io n o f t h e m a c r o p h a g e - a c t i v a t i n g e f f e c t o f t h e l a t te r w a s o b s e rv e d .
H o w e v e r , a s u p p r e s s i v e f a c t o r s e e m e d t o b e p r e s e n t i n t h e u n p u r i f i e d L K
p r e p a r a t i o n s w h e n t h e y w e r e u s e d a t h i g h c o n c e n t r a t io n s . T h e n a t u r e o f su c h a
s u p p re s s iv e f a c t o r a n d i ts po s s ib l e ro l e i n a n e rg i c s t a t e s wa r r a n t fu r t h e r s t u d y .
A s e c o n d p o ss ib l e e x p l a n a t io n f o r t h e r e l a ti v e p o t e n c y o f r e c o m b i n a n t I F N 3,
i s t h a t t h e r e c o u l d b e a c o s t i m u l a t o r c o n t a m i n a t i n g i t, s u c h a s b a c t e r i a l L P S (4 8) .
H o w e v e r , i n m i x i n g e x p e r i m e n t s , n o c o s t i m u l a t o r a c t i v i t y c o u l d b e d e m o n -
s t r a te d . F u r t h e r m o r e , w e h a v e n o t b e e n ab l e t o e n h a n c e h u m a n m a c r o p h a g e
H202 r e l e a s e w i t h L P S , u s i n g t h e s a m e c u l t u r e c o n d i t i o n s a s i n t h e p r e s e n t w o rk
(C . Na t h a n , u n p u b l i s h e d o b s e rv a t i o n s ) . F i n a l l y , n o L P S wa s d e t e c t a b l e i n t h e
p u r i f i e d r e c o m b i n a n t I F N y b y t h e l i m u l u s a m o e b o c y t e l y s a t e t e s t ( p e r s o n a l
c o m m u n i c a t i o n , D r . C . S e v a s to p o u lo s ) ( li m i t o f d e t e c t i o n , 0 . 0 5 n g / m l ) . T h i s
p re p a ra t i o n wa s e f f e c t i v e a f t e r a d i l u t io n o f m o r e t h a n 1 0 fol d. E f f e c t i v e n e s s
a f t e r s u c h d i l u t i o n v i r t u a l l y ru l e s o u t a c o n t r i b u t i o n b y t r a c e s o f L P S i n t h e
I F N o r b y a n y o t h e r m i n o r c o n t a m i n a nt .
T h u s , a t h i rd p o s si b il i ty m u s t b e c o n s i d e re d , t h a t o n e m o l e c u l e o f r e c o m b i n a n t
I F N ~ , m a y e i t h e r h a v e m o r e p o t e n t M A F a n d / o r l e s s p o t e n t a n t i v i r a l a c t i v i t y
t h a n o n e m o l e c u l e o f n a t iv e I F N y , p e r h a p s b e c a u s e o f d i f f e r e n c e s i n p o st tr a n s la -
t i o n a l m o d i f i c a t i o n s . I n f a c t , o n a p ro t e i n b a s i s , S E A- IF Ny a n d r e c o m b i n a n t
1 F N3 ' h a d s i m i l a r M AF a c t i v i t y . T h e re i s p r e c e d e n t fo r v a r i a t i o n s i n r a t i o s o f
d i f f e r e n t I F N e f f e c t s w h e n c o m p a r i n g n a t u r a l a n d r e c o m b i n a n t I F N s ( 49 ). T h e
re a s o n s fo r t h e s e v a r i a t i o n s a r e n o t we l l u n d e r s t o o d .
T h e r e h a v e b e e n a l m o s t n o p re v i o u s r e p o r t s o n t h e e f fe c t s o f I F N o n m a c r o -
p h a g e o x i d a t i v e m e t a b o l i s m . B o ra s c h i e t a l . ( 5 0 ) r e p o r t e d t h a t IF N/ 3 i n h i b i t e d
t h e s u p e r o x i d e - r e l e a s i n g c a p a c it y o f m o u s e p e r i t o n e a l m a c r o p h a g e s . H o w e v e r ,
p u r e IF N/3 wa s n o t u s e d . I t w il l b e o f i n t e r e s t t o d e t e rm i n e t h e r e l a t i o n b e t w e e n
t h e i n h i b i t o ry a c t i v i t y i n t h a t s t u d y a n d t h e f a c t o r s u p p re s s i n g m a c ro p h a g e
o x i d a ti v e m e t a b o l is m t h a t w a s d e t e c t e d i n m e d i u m c o n d i t i o n e d b y a v a r i et y o f
c e l l t y p e s , i n c l u d i n g f i b ro b l a s t s (4 6 ) . I n c o n t r a s t , n u m e ro u s r e p o r t s h a v e s u g -
g e s t e d t h a t IF N m a y e n h a n c e a n t i m i c ro b i a l a c t i v i ty o f a v a r i e t y o f c el ls a g a in s t
p a t h o g e n s o t h e r t h a n v i ru s e s , i n c l u d i n g T gondii (5 1 -5 4 ) , m a l a r i a (5 5) , Rickettsia
(56 ) , sh ige l lae (57) , sa lm onel la e (58 ) , s t aphy lococc i (59 ), and my cob ac te r ia (60 ).
T h e c a p a c it y o f h o st l y m p h o c y t e s t o s e c r e t e I F N y m o u n t s i n p a r al le l w i t h
o t h e r m a n i f e s t a ti o n s o f d e l a y e d - t y p e h y p e r s e n s it i v it y a n d c e l l - m e d i a te d i m m u n i t y
(3 4 , 3 9 , 6 1 , 6 2 ) . T h i s i s c o n s i s t e n t w i t h t h e e v i d e n c e p re s e n t e d h e re i n s u p p o r t
o f t h e h y p o th e s is t h a t I F N y m e d i a t e s m a c r o p h a g e a c t i va t i on d u r i n g t h e l a t t e r
r e s po n s e s. T h u s i t s e e m s a p p r o p r i a t e t o a s k w h e t h e r p r o v is i on o f I F N y m i g h t
f a v o ra b l y a f f e c t t h e c o u r s e o f d is e a s e s i n wh i c h p e r s i s t e n t p a ra s i t i z a ti o n o f
m a c r o p h a g e s i s a p r o m i n e n t f e a t u r e . T h e s e i n c l u d e s o m e o f t h e m o s t p r e v a l e n t
c h ro n i c i n f e c t i o n s , s u c h a s t u b e rc u l o s i s , l e p r o m a t o u s l e p ro s y , a n d l e is h m a n ia s i s.
S u m m a r y
H u m a n b l o o d m o n o n u c l e a r l e u k o c y t e s s t im u l a t e d w i th t o x o p l a s m a a n t ig e n ,
c o n c a n a v a l i n A , m e z e re i n p l u s l e n t i l l e c t i n , o r s t a p h y l o c o c c a l e n t e ro t o x i n A
s e c r e t e d a f a c t o r ( m a c r o p h a g e - a c t i v a ti n g f a c t o r, o r M A F ) t h a t e n h a n c e d t h e
c a p a c i t y o f h u m a n m a c ro p h a g e s t o r e le a s e H~ O2 a n d t o ki ll to x o p l a s m a s . T h e
s a m e ly m p h o i d s u p e r na t a n ts c o n t a i n e d I F N ~ b u t n o t I F N a o r I F NB . T h e M A r
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a c t iv i ty o f si x o f s e v e n u n f r a c t i o n a t e d s u p e r n a t a n t s w a s c o m p l e t e l y e l im i n a t e d b y
a m o n o c l o n a l a n t i b o d y t h a t n e u t r a l i z e s I F N % a n d M A F i n t h e r e m a i n i n g
s u p e r n a t a n t w a s a l m o s t c o m p l e t e l y n e u t r a l i z e d . N a t i v e IF N 3 ' p a rt i a ll y p u r i f i e d
b y tw o i n d e p e n d e n t p r o t o c o l s t o s p e c i f ic a c t i v it i e s o f 1 x 1 06 a n d 1 07 U / r a g
p r o t e i n w a s e n r i c h e d i n M A F a c t i v it y a t l e a s t a s m u c h a s i n a n t i v ir a l a c t i v it y . T h e
c a p a c i ty o f m a c r o p h a g e s t o s e c r e t e H z O u a f t e r i n c u b a t i o n i n p ar t ia l ly p u r i f ie d
n a t i v e I F N 3 , ( m e a n p e a k s t i m u l a t i o n , 8 . 8 - f o ld ) w a s g r e a t e r t h a n w i t h u n p u r i f i e d
] y m p h o k i n e s ( 3 . 8 -f o ld ) a n d s o m e t i m e s e q u a l e d o r e x c e e d e d t h e c a p a c i t y o f fr e s h ly
h a r v e s t e d m o n o c y t e s . T h e M A F a c t iv i ty o f th e p a r t ia l ly p u r i f i e d n a t i v e I F N ~ ,
p r e p a r a t i o n s w a s a b o l is h e d b y m o n o c l o n a l a n t i- I FN 3 , . F i na l ly , I F N 3 , o f > 9 9 %
e s t i m a t e d p u r i t y w a s i s ol a te d ( a t G e n e n t e c h , I n c. ) f r o m b a c t e r i a t r a n s f o r m e d
w i t h t h e c l o n e d h u m a n g e n e f o r th i s l y m p h o k i n e . R e c o m b i n a n t I F N 3 , h a d p o t e n t
M A F a c t iv i ty , s t im u l a t i n g t h e p e r o x i d e - r e l e a s in g c a p a c i ty o f m a c r o p h a g e s a n
a v e r a g e o f 1 9 . 8 - f o l d a t p e a k r e s p o n s e a n d e n h a n c i n g t h e i r a b i l i t y t o k i l l t o x o -
p l a s m a s f r o m 2 . 6 + 1 . 3 % f o r u n t r e a t e d c e l ls t o 5 4 + 0 . 4 % f o r t r e a t e d c e ll s.
A t t a i n m e n t o f 5 0 % o f th e m a x i m a l e l e v a t i o n in p e r o x i d e - r e le a s i n g c a p a c i t y
r e q u i r e d a g e o m e t r i c m e a n c o n c e n t r a t i o n o f 0 .1 a n ti v ir a l U / m ] o f re c o m b i n a n t
I F N 3 , , w h i c h i s e s t i m a t e d t o b e - ~ 6 p i c o m o l a r f o r t h is p r e p a r a t i o n . P e r o x i d e
s e c r e t o r y c a p a c i ty a n d t o x o p l a s m a c i d a l a c t iv i ty o f m a c r o p h a g e s p e a k e d 2 - 4 d
a f t e r e x p o s u r e t o I F N 3 , . P e r o x i d e - s e c r e t o r y c a p a c i t y r e m a i n e d e l e v a t e d d u r i n g
a t l e as t 6 d o f c o n t i n u o u s e x p o s u r e , b u t t h e e f f e c t o f I F N 3, w a s r e v e r s e d w i t h in
a b o u t 3 d o f i ts r e m o v a l . A c t i v a t i o n w a s u s u a ll y b u t n o t i n v a r i a b l y a c c o m p a n i e d
b y c h a r a c t e r i s t i c c h a n g e s i n c e ll m o r p h o l o g y . T h u s , I F N 3 , a c t i v a t e s h u m a n
m a c r o p h a g e o x i d a t iv e m e t a b o l i s m a n d a n t i m i c ro b i a l a c ti v it y , a n d a p p e a r e d t o b e
t h e o n l y f a c t o r c o n s i s te n t ly c a p a b l e o f d o i n g s o i n th e d i v e r s e L K p r e p a r a t i o n s
t e s t e d .
Zanvil Cohn i s warmly acknow ledged for encou raging us to unde r take these s tudies a nd
for his cri t ical advice and review. We thank Genentecb, Inc., in part icular Sang-He Lee
and Costa Sevastopoulos, for thei r ge nerous provision o f recombinant IFN~ , and for
helpful suggestions, M . Chang fo r purification of MLL-IFN3,, G. Spitalny and N . Paw-
lowski for advice, and R. Sch reiber and J . Vi l~ek for com municat ing result s p r ior to
publication. W e are gratefu l to Na ncy DeSantis, Denise Cartell i , Sylvia An derson , Carol
Deboer , Tobie Overdank, and Richard Bonomo for thei r ski l l fu l assis tance, and Judy
Adam s and Ann a Szuro-Sudol for aid in f igure preparat ion.
Received or publication 4 May 1983
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