J Exp Med-1983-Nathan-670-89

7/18/2019 J Exp Med-1983-Nathan-670-89

1/20

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

7/18/2019 J Exp Med-1983-Nathan-670-89

2/20

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

7/18/2019 J Exp Med-1983-Nathan-670-89

3/20

6 7 2

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


7/18/2019 J Exp Med-1983-Nathan-670-89

4/20

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


7/18/2019 J Exp Med-1983-Nathan-670-89

5/20

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


7/18/2019 J Exp Med-1983-Nathan-670-89

6/20

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).


7/18/2019 J Exp Med-1983-Nathan-670-89

7/20

6 7 6

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 .


7/18/2019 J Exp Med-1983-Nathan-670-89

8/20

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 -


7/18/2019 J Exp Med-1983-Nathan-670-89

9/20

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.


7/18/2019 J Exp Med-1983-Nathan-670-89

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


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


7/18/2019 J Exp Med-1983-Nathan-670-89

13/20

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


7/18/2019 J Exp Med-1983-Nathan-670-89

14/20

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


7/18/2019 J Exp Med-1983-Nathan-670-89

15/20

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


7/18/2019 J Exp Med-1983-Nathan-670-89

16/20

NATHAN ET AL 685

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

R e f e r e n c e s

1. Mackaness, G. B. 1969. Th e influence of imm unologically co m mitted lymphoid cells

on macrophage activity in vivo.

J Ex p Me d

129:973.

2. Mackaness, G. B. 1970. T he mechanism of ma croph age activation. In Infectious

Agents and Host Reactions. S. Mudd, editor. W. B. Saunders Co., Philadelphia, 61-

75.

3. Na than, C. F., H. G. Rem old, an dJ. R. David. 1973. Characterization of a lymp hocy te

factor which al ters macrophage funct ions.J .

Ex p Me d

137:275.

4. Na than, C. F., M. L. Karnovsky, an dJ . R . David. 1971. Alterations o f m acro phag e

functions by mediators from lymphocytes.

J Exp Med

33:1356.

5. K rahen buhl, J. L., and J. S. Rem ington. 1971. In vitro induc tion of nonspecific


7/18/2019 J Exp Med-1983-Nathan-670-89

17/20

6 8 6

INTERFERON-~ AS M ACROPHAGE-ACTIVAT1NG FACTOR

r es i s tance i n m acr oph ages by spec if i ca l ly s ens it i zed l ym phocy t es . Infect . hnmun. 4: 337 .

6 . F owles R . E . I . M . F a j a r do J . L . L e i bowi t ch a nd J . R . Dav i d . 1973 . T h e enhance -

me nt o f mac r oph age bac t e r ios t a s is by p r oduc t s o f ac t iva t ed l ymphocy t es .J . Exp . M ed .

138: 952 .

7 . Bor ges J . S . and W . D . John son J r . 1975 . I nh i b i t ion o f mul t i p l i ca ti on o f Toxoplasma

gondii

b y h u m a n m o n o c y t e s e x p o s e d to T l y m p h o c y t e p r o d u c t s . J .

Exp. Med.

141: 483 .

8 . A nder so n S . E . S . Bau t i st a and J . S . Rem i ng t on . 1976 . I nduc t i on o f r e s i s tance to

Toxoplasma gondii i n h u m a n m a c r o p h a g e s b y s o l u b le l y m p h o c y t e p r o d u c t s . J . Immunol .

117: 381 .

9 . Nog ue i r a N . and Z . A . Cohn . 1978 .

Trypanosoma cruzi:

i n v i t r o i nduc t i on o f

mac r oph age mi c r ob i c i da l ac t iv i t y .J. Exp. Med. 148: 288 .

10 . Na t han C . F . N . Nogue i r a C . Juang bhan i ch J . El li s and Z . A . Cohn . 1979 .

A c t iv a t io n o f m a c r o p h a g e s i n v i v o a n d i n v i tr o . C o r r e l a t i o n b e t w e e n h y d r o g e n

per o x i de r e l ease and k i ll ing o f

T~Tpanosoma cruzi. J . Exp. Med.

149: 1056 .

1 1. M u r r a y H . W . a n d Z . A . C o h n . 1 9 8 0 . M a c r o p h a g e o x y g e n - d e p e n d e n t a n t im i c r o b ia l

ac t iv i t y . I I I . E nh anced ox i da t i ve me t abo l i sm as an ex pr es s i on o f macr o phag e ac t iva -

t i on . J . Exp . Med . 152: 1596 .

12 . B f i chmul l e r Y . and J . M aue l . 1981 . S t ud i es on t he mechan i sms o f mac r opha ge

ac t i va ti on : pos s ib l e i nvo l vem ent o f oxy gen me t abo l i t e s i n k i l li ng o f

Leishma nia en rietti

b y a c t iv a t e d m o u s e m a c r o p h a g e s . J. Reticuloendothel. Sac. 29: 181 .

13. W alker L . a nd D. B. Low rie . 1981. Ki l ling ofMycobacterium microt i b y i m m u n o l o g i -

ca ll y ac t i va t ed m acr ophages . Nature Lond . ) . 293: 69 .

14 . Nakagaw ar a A . N . M . DeS an t is N . Nog ue i r a and C . F . Na t han . 1982 . L ymph oki nes

e n h a n c e t h e c a p a c i ty o f h u m a n m o n o c y t e s t o s e c r e te r e a c t iv e o x y g e n i n t er m e d i a t e s.

J. Clin. Invest. 7 0 : 1 0 4 2 .

15. Mu rray H. W. and D. M. Car te l l i . 1983. Ki l l ing of int racel lu lar Leishmania donovani

b y h u m a n m o n o n u c l e a r p h a g o c y t es : e v i d e n c e f o r o x y g e n - d e p e n d e n t a n d - i n d e p e n d -

ent le i shmanicidal act ivi ty. J. cI in . Invest . 72: 32 .

16 . M ur r ay H . W . G . I . Byr ne C . D . Rot he r me l and D . M . Car t e ll i . 1983 . L ym pho ki ne

enhance s oxy gen - i ndep end en t ac t i v it y aga i ns t i n t r ace l lu l a r pa t hogens . J. Exp. Med.

In press .

17 . Na t han C . F. 1983 . M echan i sms o f ma cr oph age an t i mi c r ob i a l ac t iv i t y Trans. R. Soc.

Trop. Med. Hyg. In press .

1 8 . S t e eg P . S . R . N . M o o r e H . M . J o h n s o n a n d J . J . O p p e n h e i m . 1 9 8 2 . R e g u l a t io n o f

m u r i n e m a c r o p h a g e I a a n t i g e n e x p r e ss i o n b y a l y m p h o k i n e w i th i m m u n e i n t e r f e r o n

ac t i v i t y . J . Exp . Med . 156: 1780 .

19 . B asham T . Y . a nd T . C . M er i gan . 1983 . Recom bi nan t i n t e r f e r on - y i nc r eases HL A-

DR syn t hesi s and expr es s i on . J . l mm u n o l . 130: 1492 .

20. Pac e J . L . S . W. Russell B. A. To rre s H. M. Joh nso n and P . W. Gray . 1983.

R e c o m b i n a n t m o u s e ~ i n t e r f e r o n i n d u c e s t h e p r i m i n g s t e p in m a c r o p h a g e a c t i va t io n

for t um or cel l kil ling. J . hnmuno l . 130: 2011 .

21. Schr ieb er R. D. J . L . Pace S . W. Russell A. Al tm an and D. H. Katz . 1983.

M a c r o p h a g e a c t iv a t in g f a c t o r p r o d u c e d b y a T c e ll h y b r i d o m a : m o l e c u l a r e v i d e n c e

f or i ts i den t i t y w i t h gamm a i n t e r f e r on . J . hnmuno l . In press .

22 . Hovi T . O . S aksel a and A . Vaher i . 1981 . I nc r eased sec r e ti on o f p l a smi nogen

a c t i v a to r b y h u m a n m a c r o p h a g e s a f t e r e x p o s u r e t o l e u k o c y t e in t e r f e r o n .

FEBS Fed .

Eur. Biochem. Sot .) Lef t . 129: 233 .

23 . Jones C . M . L . Var es i o R . H . Her b e r m an and S . P es t ka . 1982 . I n t e r f e r on act i va te s

m a c r o p h a g e s t o p r o d u c e p l a s m i n o g en a c t i v a to r .

J. Interferon Res.

2: 377 .

24 . Ha mb ur g S . I . H . B . F l e it J . C . Unke l es s and M . Rab i nov i t ch . 1980 . M o non uc l ea r

phagocy t es : r e sponde r s t o and p r od uce r s o f i n t e r f e r on . Ann. N . Y.

Acad. Sci .

350: 72 .


7/18/2019 J Exp Med-1983-Nathan-670-89

18/20

N A T H A N E T A L . 6 8 7

2 5 . G u y r e , P . M . , P . M . M o rg an e l l i , an d R . M i l le r . 1 9 8 3 . A u g m en ta t i o n o f h u m a n

m o n o c y t e F c g a m m a r e c e p t o r s b y r e c o m b i n a n t g a m m a ( i m m u n e ) i n t e r f e r o n . Fed.

Proc. 42 :428 . (Abst r . ) .

2 6 . G ray , P . W . , D . W . L eu n g , D . P en n ica , E . Y e lv e r to n , R . N a ja r i an , C . C . S im o n sen ,

R . D ery n ck , P . J . S h e rw o o d , D . M . W al l ace , S . L . B e rg e r , A . D . L ev in so n , an d D . V .

G o e d d e l . 1 9 8 2. E x p r e s s i o n o f h u m a n i m m u n e i n t e r f e r o n c D N A i n

E . co l i

a n d m o n k e y

cells. N a t u r e L o n d . ) . 2 9 5 :5 0 3 .

2 7 . N a k a g a w a r a , A ., C . F . N a t h a n , a n d Z . A . C o h n . 1 9 8 1 . H y d r o g e n p e r o x i d e m e t a b o l i s m

i n h u m a n m o n o c y t e s d u r i n g d i f f e r e n t i a ti o n i n v i tr o .

J. Clin . Invest .

6 8 :1 2 4 3 .

2 8 . L o w ry , O . H . , N . J . R o seb r o u g h , A . L . F a r r , an d R . J . R an d a l l . 1 9 5 1 . P ro t e in

m e a s u r e m e n t w i t h t h e F o l i n p h e n o l r e a g e n t . J. Biol . Chem. 193 :265 .

2 9 . M u r ray , H . W . , C . W . Ju an g b h a n ich , C . F . N a th an , an d Z . A . C o h n . 1 9 7 9 . M acro -

p h a g e o x y g e n - d e p e n d e n t a n t i m i c r o b i a l a c ti v it y . II . T h e r o l e o f o x y g e n i n t e r m e d i a t e s .

J . E x p . M e d . 1 5 0 :9 5 0 .

30 . Rub in , B . Y. , A . H. Ba r ta l , S . L . Ande rso n , S . K . Mil le t , Y . Hirsha u t , an d C . Fei t .

1 9 83 . T h e a n t i c e ll u l a r a n d p r o t e i n - i n d u c i n g a c t iv i ti e s o f h u m a n 3 ' i n t e r f e r o n p r e p a -

r a t i o n s a r e m e d i a t e d b y t h e i n t e r f e r o n . J . h n m u n o l . 1 3 0 :1 0 1 9 .

3 1 . R u b in , B . Y ., an d S . L. G u p ta . 1 9 8 0 . D i f f e r en t i a l e f fi cac i e s o f h u m an ty p e I an d ty p e

I I i n t e r f e ro n s a s an t iv i r a l an d an t ip ro l i f e r a t i v e ag en t s . P roc . Na t l . A cad . S c i . USA .

7 7 :5 9 2 8 .

3 2 . S t ew ar t , W . E ., I I I , ed i t o r . 1 9 8 1 . T h e In t e r f e ro n S y s t em . 2 n d ed . S p r in g e r -V e r l ag ,

N e w Y o r k . 1 3 - 2 6 .

3 3 . W h ee lo ck , E . F . 1 9 6 5 . In t e r f e ro n - l i k e v i ru s - in h ib i t o r i n d u ced in h u m an l eu k o cy tes

b y p h y t o h e m a g g l u t i n i n . Sc ience Wa sh . DC ) . 149 :310 .

3 4 . G re en , J . A . , S . R . C o o p e rb an d , an d S . K ib r i ck. 1 9 6 9 . Im m u n e sp ec if i c i n d u c t io n o f

i n t e r f e r o n p r o d u c t i o n i n c u l t u r e s o f h u m a n b l o o d l y m p h o c y t e s . Sc ience Wash . DC ) .

1 6 4 :1 4 1 5 .

3 5 . Y ip , Y . K ., R . H . L . P an g , J . D . O p p en h e im , M . S . N a ch b ar , D . H en r ik sen , I .

Z e r e b e c k y j - E c k h a r d t , a n d J . V il ~z ek . 1 9 81 . S t i m u l a t i o n o f h u m a n g a m m a i n t e r f e r o n

p r o d u c t i o n b y d i t e r p e n e e s t er s .

In fec t . hnmun .

3 4 :1 3 1 .

3 6 . L o ck s l ey , R , M . , C . B . W i l so n , an d S . J . K leb a n o f f . 1 9 8 2 . R o le fo r en d o g en o u s an d

a c q u i r e d p e r o x i d a s e i n t h e t o x o p l a s m a c i d a l a c t iv i ty o f m u r i n e a n d h u m a n m o n o n u -

c l ea r p h ag o cy tes .

J. Clin. Invest.

6 9 :1 0 9 9 .

3 7 . L ee , S . H . S . , an d L . B . E p s t e in . 1 9 8 0 . R ev e r s ib l e i n h ib it i o n b y i n t e r f e ro n o f t h e

m a t u r a t i o n o f h u m a n p e r i p h e r a l b l o o d m o n o c y t e s t o m a c r o p h a g e s . Cel l . Immuno l .

5 0 :1 7 7 .

3 8 . H o v i , T . , O . S ak se la , K . L in n a v u k o r i , A . V ah er i , an d K . C an te l l. 1 9 8 1 . A c t iv a t i o n o f

m o n o c y t e f u n c t i o n s b y i n t e r f e r o n s . In T h e B i o lo g y o f t h e I n t e r f e r o n S y s t em . E . D e

M a e y e r , G . G a l a s s o, a n d H . S c h e l le k e n s , ed i t or s . E l s e v i e r / N o r t h - H o l l a n d , N e w Y o r k .

2 1 7 - 2 2 0 .

3 9 . H av e lL E . A . , G . L . S p i t a ln y , an d P . J . P a t e l . 1 9 8 2 . E n h an ce d p ro d u c t io n o f m u r in e

in t e rf e ro n ~ b y T ce l l s g en e ra t ed i n r e sp o n se t o b ac t e r i a l in f ec t i o n . J . E x p . M e d .

156 :112 .

4 0 . K asah a ra , T . , J . J . H o o k s , S . F . D o u g h er ty , an d J . J . O p p en h e im . 1 9 8 3 . In t e r l e u k in

2 - m e d i a t e d i m m u n e i n t e r f e r o n ( I F N - ' r ) p r o d u c t i o n b y h u m a n T c e ll s a n d T c e ll

subsets .

J . h n m u n o l .

1 3 0 :1 7 8 4 .

4 1 . G r a y , P . W . , a n d D . V . G o e d d e l . 1 9 8 2 . S t r u c t u r e o f t h e h u m a n i m m u n e i n t e r f e r o n

g en e . N a t u r e L a n d . ) . 2 9 8 :8 5 9 .

4 2 . D e v o s , R . , H . C h e r o u t r e , Y . T a y a , W . D e g r a v e , H . V a n H e u v e r s w y n , a n d W . F i er s .

1 9 8 2. M o l e c u l a r c lo n i n g o f h u m a n i n t e r f e r o n c D N A a n d i ts e x p r e s s i o n in e u k a r y o t i c

cells.

N u d . A c i d s R e s .

10 :2487 .


7/18/2019 J Exp Med-1983-Nathan-670-89

19/20

68 8 INTERFERON-3 , AS MACROPHA GE-ACTIVATING FACTOR

43. Vi l~ek , J . , K . T . Pear ls te in , J . Le, D. S . S tone -W olff , H . C . Kelke r , and Y. K. Yip .

1 9 83 . I n t e r f e r o n - g a m m a : i nt e r a c t i on s w i t h o t h e r l y m p h o k i n e s . In T h e B i o l og y o f t h e

I n t e r f e r o n S y s t e m . H . S c h e l l e k e n s , e d i t o r . E l s e v i e r / N o r t h - H o l l a n d , N e w Y o r k . I n

press .

4 4 . P i l l em er , E ., an d I . L . W e i s sm an . 1 9 8 1 . A m o n o c lo n a l an t ib o d y th a t d e t ec t s a V k-

T E P C 1 5 i d i o t yp i c d e t e r m i n a n t c r o s s -r e a c t iv e w i th a T h y - 1 d e t e r m i n a n t . J. Exp. Med.

1 5 3 :1 0 6 8 .

4 5 . M iz rah i , A . , J . A . O M al l ey , W . A . C a r t e r , A . T ak a t su k i , G . T am u ra , an d E .

S u lk o w sk i. 1 9 7 8 . G ly co sy la t io n o f i n t e r f e ro n s . E f fec t s o f t u n i cam y c in o n h u m a n

i m m u n e i n t e r f e r o n . J. Biol. Chem. 2 5 3 :7 6 1 2 .

4 6 . S zu ro -S u d o l , A . , an d C . F . N a th a n . 1 9 8 2 . S u p p ress io n o f m a cro p h ag e o x id a t iv e

m e t a b o l i s m b y p r o d u c t s o f m a l i g n a n t a n d n o n m a l i g n a n t c el ls . J . Exp . Med. 156 :945 .

4 7 . F l e i sch m an , , W . R . , J r . , J . A . G e o rg iad e s , L . C . O sb o rn e , F . D ian zan i , an d H . M .

J o h n s o n . 1 9 7 9. I n d u c t i o n o f a n i n h i b it o r o f i n t e r f e r o n a c t i on i n a m o u s e l y m p h o k i n e

p r e p a r a t i o n . Infect. Imm un. 2 6 :9 4 9 .

4 8 . P ab s t , M . J . , H . B . H e d eg aa rd , an d R . B . Jo h n s to n , J r . 1 9 8 2 . C u l tu r ed h u m a n

m o n o c y t e s r e q u i r e e x p o s u r e t o b a c t e r i a l p r o d u c t s t o m a i n t a i n a n o p t im a l o x y g e n

rad ica l r e sp o n se . J . I mmu ~o l . 128 :123 .

4 9 . S t eb b in g , N . , P. K . W eck , J . T . F en n o , S . A p p er so n , an d S . H . L ee . 1 9 8 1 . C o m p a r i so n

o f th e b i o lo g i ca l p r o p e r t i e s o f n a t u r a l a n d r e c o m b i n a n t D N A d e r i v e d h u m a n i n t e r -

f e ro n s . In T h e B io lo g y o f t h e In t e r f e ro n S y s t em . E . D e M ae y er , G . G a las so , an d H .

S c h e ll e k en s , e d it o r s . E l s e v i e r / N o r t h - H o l l a n d , N e w Y o r k . 2 5 - 3 3 .

5 0 . B o rasch i , D ., P . G h ezz i , M . S a lm o n a , an d A . T ag l i ab u e . 1 9 8 2 . IF N - /3 -in d u ced r ed u c -

t i on o f s u p e r o x i d e a n i o n g e n e r a t i o n b y m a c r o p h a g e s . Immunology 4 5 :6 2 1 .

5 1 . R em in g to n , J . S ., an d T . C . M er ig an . 1 9 6 8 . In t e r f e ro n : p ro t ec t io n o f ce l ls i n fec t ed

w i t h a n i n t r a c e l l u la r p r o t o z o a n Toxoplasma gondii) . Science W ash. DC ). 161 :804 .

5 2 . S h i r a h a ta , T . , a n d K . S h i m i zu . 1 9 8 0 . P r o d u c t i o n a n d p r o p e r t i e s o f i m m u n e i n t e r f e r o n

f ro m sp l een ce l l cu l tu res o f t o x o p lasm a- in fec t ed m ice . Microbiol. Imm unol. 2 4 :1 1 0 9 .

5 3 . S ak u ra i , H . , Y . T ak e i , Y . O m ata , an d N . S u zu k i. 1 9 8 1 . P ro d u c t io n an d p ro p e r t i e s o f

t o x o p l a s m a g r o w t h i n h i b it o r y f a c t o r ( T o x o - G I F ) a n d i n t e r f e r o n ( I F N ) i n t h e l y m p h o -

k in es an d th e c i r cu l a t i o n o f t o x o p lasm a im m u n e m ice . Z b l. B ak t . H y g I . A b t . O r ig .

A . 2 5 1 :1 3 4 .

5 4 . P f e f f e r k o r n , E . R . , a n d P . M . G u y r e . 1 9 8 3 . R e c o m b i n a n t h u m a n g a m m a i n t e r f e r o n

b lo ck s t h e g ro w th o f Toxoplasma gondii i n cu l tu red h u m an f ib ro b las t s . Fed. Proc.

42 :964 . (Abstr . ) .

5 5 . J ah i e l , R . I . , J . V i l cek , an d R . S . N u ssen zw e ig . 1 9 7 0 . E x o g en o u s in t e r f e ro n p ro t ec t s

m ice ag a in s t Plasmodium berghei m ala r i a . Nature Lond.) . 2 2 7 :1 3 5 0 .

5 6 . K aza r , J . , P . A . K rau tw u rs t , an d F . B . G o rd o n . 1 9 7 1 . E f fec t o f i n t e r f e ro n an d

in t e r f e ro n in d u ce r s o n in fec t io n s w i th a n o n v i r a l i n t r ace l lu l a r m ic ro o rg an i sm ,

Rick-

ettsia akari. Infect. Immun. 3 :8 1 9 .

5 7 . G o b er , L . L . , A . E . F r i ed m an -K ien , E . A . H av e l l , an d J . V i l~ zek . 1 9 7 2 . S u p p ress io n

o f t h e i n t r a c e l lu l a r g r o w t h o f

Shigella flexneri

i n ce l l cu l tu res b y in t e r f e ro n p rep a ra -

t ions and by po ly inosin ic-po lycy t id i l ic acid . Infect. Immun. 5 :370 .

5 8 . l zad k h ah , Z . , A . D . M an d e l , an d G . S o n n en fe ld . 1 9 8 0 . E f fec t o f t r ea tm en t o f m ice

w i t h s e r a c o n t a i n i n g g a m m a i n t e r f e r o n o n t h e c o u r s e o f i n f e c t i o n w i t h

Salmonella

typhimurium s t r a in L T -2 . J. bTterferon Res. 1 :137 .

5 9 . Im an i sh i , J . , K . O i sh i , A . S u g an u m a , Y . Y o k o ta , an d T . K i sh id a . E n h an c em en t o f

b a c t e ri c i d a l a c t iv i ty 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 a g a i n s t Staphylococcus aureu s b y

m o u s e i n t e r f e r o n p r e p a r a t i o n s . BikenJ . 25 :71 .

6 0 . M izu n o e , K . , M . H i rak i , Y . N ag an o , an d N . M a eh ara . 1 9 7 5 . S u p p ress io n o f i n t r ace l -

lu l a r m u l t i p l i ca t io n o f

Mycobacterium tuberculosis

b y v i ru s - in h ib i t i n g f ac to r o r i n t e r -

f e ro n . Jpn . j. Microbiol. 19 :235 .


7/18/2019 J Exp Med-1983-Nathan-670-89

20/20

N A T H A N E T A L . 6 8 9

6 1 . G lasg o w L . A . 1 9 6 6 . L eu k o cy te s an d in t e r f e ro n in t h e h o s t r e sp o n se to v i r a l

i n f ec t i on s . I I . E n h a n c e d i n t e r f e r o n r e s p o n s e o f l e u k o c y te s f r o m i m m u n e a n i m a l s. J .

B a c t e r i o l 9 1 : 2 1 8 5

6 2 . S a lv in S . B . J . S . Y o u n g n e r an d W . H . L ed e re r . 1 9 7 3 . M ig ra t io n in h ib i to ry f ac to r

an d in t e r f e ro n in t h e c i r cu l a t i o n o f m ice w i th d e l ay ed h y p er sen s i t i v i t y . In fec t lmmun

7 :68 .


J Exp Med-1983-Nathan-670-89

Documents

Transcript of J Exp Med-1983-Nathan-670-89