Ontogeny of ‘macrophage’ function. VI. Down-regulation for Ia-expression of newborn mouse macrophages by endogenous β -interferon

DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. 12, pp. 645-655, 0145-305X/88 $3.00 + .00 Printed in the USA Copyright (c) 1988 Pergamon Press plc All rights reserved

1988

ONTOGENY O F 'MACROPHAGE' FUNCTION. VI. D O W N - R E G U L A T I O N F O R I a - E X P R E S S I O N O F N E W B O R N M O O S E M A C R O P H A G E S B Y E N D O G E N O U S ~-IhTzI~-zKON.

Makoto Kitaura, Takuma Ka~o, Kayo Inaba, Yoshihiko Watanabe, Yoshimi Kawade and Shigeru Muramatsu # Department of Zoology~ Faculty of Science and The Institute for Virus Research, Kyoto University, Kyoto, 606 Japan

ABSTRACT

Peritoneal exudate macrophages (M~) of newborn mice (NB-M~) were a p p a r e n t l y a l m o s t i n c a p a b l e of e x p r e s s i n g Ia a n t i g e n e v e n if stimulated by IFN-y. No significant difference was observed in the number and the affinity of receptors for IFN-y between NB-M~ and M @ of a d u l t mice (Ad-M~). A d d i t i o n of i n d o m e t h a c i n , a prostaglandin synthesis inhibitor, was ineffective in enhancing the Ia-expression of NB-M~. Responsiveness of NB-M~ to IFN-~, however, was disclosed by the addition to the culture of antiIFN- 8 or a n t i - I F N - ~ /8, b u t n o t anti-IFN- ~ antibody. R e s p o n s i v e n e s s of N B - M @ to I F N - y was not i m p r o v e d by the depletion of fibroblasts from NB-M~ populations. These results strongly argue that Ia-expression of NB-M~, which is otherwise to be induced by IFN-y , is suppressed by IFN-8 derived from NB-M~ themse ives.

INTRODUCTION

We have reported in this series of studies that peritoneal exudate m a c r o p h a g e s (M@) of n e w b o r n mice (NB-M@) are superior to those of a d u l t s (Ad-M~) in the phagocytic activity and in the antitumor activity (1,2). On the other hand, Ia (class II MHC antigens)-bearing (Ia+) M~ in NB-M~ are v e r y few in c o m p a r i s o n with A d - M ~ (3). M o r e r e c e n t l y , we h a v e also reported that a l p h a - i n t e r f e r o n ( I F N - d ) and b e t a - i n t e r f e r o n (IFN-~ )

#

TO whom correspondence should be sent. 645

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i n h i b i t the i n d u c t i o n of I a - e x p r e s s i o n of A d - M @ by g a m m a - i n t e r f e r o n (IFN-y), and that N B - M @ appear almost unresponsive to I F N - y , but become r e s p o n s i v e to I F N - y , t h o u g h w e a k e r t h a n A d - M @ , in the p r e s e n c e of a n t i I F N - ~ a n t i b o d y (4). S n y d e r et al., h o w e v e r , r e p o r t e d t h a t p u t a t i v e M ~ p r e c u r s o r in n e o n a t a l s p l e e n c o u l d s u p p r e s s I a - e x p r e s s i o n of M ~ by r e l e a s i n g some arachidonic metabolites, p r o b a b l y p r o s t a g l a n d i n s (PGs) (5), w h i c h i n t e r f e r e w i t h s e v e r a l M ~ f u n c t i o n s (6,7,8). Moreover, IFN-~ binding ability of N B - M ~ has not been yet compared with that of Ad-M~. The present experiments were conducted to elucidate the main cause of low responsiveness of N B - M ~ to I F N - y , and to d e f i n e the c e l l type responsible for production and/or d e l i v e r y of the suppressive factor. The results show that Ia-expression of N B - M ~ is suppressed m a i n l y by IFNin an apparent autocrinical manner.

MATERIALS

AND

METHODS

Mice. C 3 H / H e S I c m i c e age 8 - 1 2 w k s ( S h i z u o k a Agricultural Cooperative A s s o c i a t i o n for L a b o r a t o r y A n i m a l s , S h i z u o k a ) w e r e u s e d as a d u l t , and those within 24hr after birth as newborn which were raised in our breeding colony. Reagents. Murine natural IFN- y , m o n o c l o n a l antibodies (mAbs) for murine I F N - ~ (4EAI) and I F N - ~ (7FD9), and a n t i - I F N - ~/8 p o l y c l o n a l antibody (mona2 4) w e r e p r e p a r e d as d e s c r i b e d p r e v i o u s l y (4). One ~ g of 4EAI and 7FD9 neutralize 103 IU of I F N - ~ and I F N - ~ , respectively. One ~ i of mona2~ neutralize I0 IU of IFN- ~/~. Recombinant murine IFN- y (rIFN-Y) with a s p e c i f i c a c t i v i t y of 107 U / m g p r o t e i n was d o n a t e d by S h i o n o g i R e s e a r c h Laboratory, Osaka. Indomethacin (IM)(Sigma Chemical Co., St. Louis, MO.) w a s u s e d as an i n h i b i t o r of p r o s t a g l a n d i n s y n t h e s i s . 10.2.16 (anti-Iak), 2 . 4 G 2 ( a n t i - F c R ) , F 4 / 8 0 ( a n t i - M ~ a n t i g e n ) and H O - 1 3 - 4 (anti-Thy-l.2) u s e d for s t a i n i n g and c y t o l y s i s w e r e p r o v i d e d by Dr. R. M. S t e i n m a n (The R o c k e f e l l e r U n i v e r s i t y , N e w York). L o w - t o x - M rabbit c o m p l e m e n t (C') was purchased from Cedarlane Laboratories Co., Ltd. (Hornby, Ontario). E n d o t o x i n (LPS) c o n t e n t s of the r e a g e n t s and the c u l t u r e m e d i u m w e r e quantified by an improved limulus test (Toxicolor test, Seikagaku Kogyo, Ltd., Tokyo). Natural I F N - y , r I F N - y , a n t i - I F N - ~ , a n t i - I F N - 8 , and anti-IFN- ~ / 8 c o n t a i n e d 0.5ng L P S / 1 0 4 U, 0.5ng L P S / 1 0 4 U, 0.04 ng L P S / I ~ g , 0.02 ng L P S / I ~ g , and 6 pg L P S / I m l , r e s p e c t i v e l y . O t h e r r e a g e n t s and the culture medium contained no detectable amounts of LPS. M@ culture and immunofluorescence. Procedures for preparing and c u l t u r i n g M ~ were described in detail p r e v i o u s l y (1,4). B r i e f l y , thioglycollate m e d i u m - e l i c i t e d peritoneal exudate c e l l s (TGC-PEC) obtained from adult and n e w b o r n m i c e 4 d a y s a f t e r an i.p. i n j e c t i o n of TGC w e r e a l l o w e d to a d h e r e onto 13 mm diameter round glass c o v e r s l i p s for 2 to 3 hr, washed to remove nonadherent cells, and treated with 20 ~g/ml Mitomycin C at 37 ° C. M~ on c o v e r s l i p s thus prepared were cultured in 16 mm dishes of 24-well plates (Nunc, R o s k i l d e ) . In some e x p e r i m e n t s , T G C - P E C b e f o r e a d h e r e n c e were treated with anti-Thy-l.2 plus C'. The adherent M~ were cultured for 6-8 d a y s in 1 ml of c u l t u r e m e d i u m (RPMI 1 6 4 0 s u p p l e m e n t e d w i t h 10% f e t a l b o v i n e s e r u m (FBS)) w i t h d i f f e r e n t r e a g e n t s . Surface antigens were v i s u a l i z e d by i n d i r e c t i m m u n o f l u o r e s c e n c e a f t e r f i x a t i o n of M ~ w i t h 1% paraformaldehyde (9). M o r e t h a n 200 c e l l s w e r e c o u n t e d to e v a l u a t e the

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percentages of fluorescence p o s i t i v e cells. S a t u r a t i o n b i n d i n g a n a l y s e s of I F N - Y to M~. R e c o m b i n a n t IFN-y was radioiodinated with 125I-Bolton-Hunter r e a g e n t (i0,ii). M ~ , prepared by a d h e r e n c e of T G C - P E C for 2hr, w e r e i n c u b a t e d in 2 4 - w e l l p l a t e (5x10 c e l l s / w e l l ) for 4hr at 20°C with increasing concentrations of 1 2 5 I - r I F N - ~ in M E M s u p p l e m e n t e d w i t h 1% g l u c o s e , 5 m M HEPES, a n d 10% FBS. C e l l s w e r e washed three times with cold MEM, lysed with 2% SDS, and the r a d i o a c t i v i t y w a s c o u n t e d . S p e c i f i c b i n d i n g was d e f i n e d by the s u b t r a c t i o n of n o n s p e c i f i c b i n d i n g o c c u r r i n g in the p r e s e n c e of 2 0 0 - f o l d e x c e s s of c o l d rIFN- ~ f r o m the t o t a l b i n d i n g . The n u m b e r of s p e c i f i c b i n d i n g sites, or receptors, and their affinity were c a l c u l a t e d by the Scatchard Analysis M e t h o d (12).

RESULTS

Apparent incompetence of NB-M~ to express Ia antigens T h i s e x p e r i m e n t was c o n d u c t e d to find a p o s s i b l e c u l t u r e c o n d i t i o n which w o u l d induce Ia-expression of N B - M ~ by IFN-~ . N B - M @ were virtually incompetent to express Ia antigen during any culture period (Fig. IA) and in c u l t u r e s c o n t a i n i n g any d o s e of I F N - y (Fig. IB). The e f f e c t of IM, which is also i l l u s t r a t e d in Fig. 1 , w i l l be described later.

A)

100 -

B'

4d culture

100

IFN-Y:12U/mt

÷

~50

I

t

I

2

4

6

tU50

0

5

IFN-

Days FIG.

E f f e c t of IFN- 7 on I a - e x p r e s s i o n (triangles). M~ were cultured with indomethacin.

15

50

150

(U/mr)

1

of Ad-M~ (circles) and NB-M@ (closed) or w i t h o u t (open) 1 )/g/ml

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Specific binding sites for I F N - y on NB-M@ There is a p o s s i b i l i t y that low responsiveness of N B - M ~ to IFN-y is due to a small number and/or low affinity of IFN-y receptors on N B - M ~ in comparison with Ad-M~. To confirm this , we analyzed the specific binding of I F N - ~ to NB-M~, and found that IFN-~ s p e c i f i c a l l y binds to N B - M ~ in a saturable manner (Fig. 2). Since N B - M ~ were c o l l e c t i b l e in a r e l a t i v e l y s m a l l n u m b e r , we d e t e r m i n e d the IFN- 7 b i n d i n g at o n l y t h r e e doses. Therefore, the v a l u e s calculated f r o m t h i s e x p e r i m e n t m a y not be d e f i n i t i v e , but t h e y can s e r v e at l e a s t as a b a s i s for c o m p a r i s o n s w i t h those in A d - M ~ which were determined more precisely. Affinity and specific binding s i t e s / c e l l c a l c u l a t e d from this N B - M ~ analysis were as follows: K a = 3 . 8 x 1 0 9 M -I and 25,000 s i t e s / c e l i. Repeated experiments under s a t u r a t i o n c o n d i t i o n , w h i c h w e r e not s u f f i c i e n t for S c a t c h a r d a n a l y s i s , a l s o showed that N B - M ~ have 25,000-26,000 specific binding sites (data not shown). In contrast, the v a l u e s of A d - M ~ in our separate experiments were K a = l . 7 x l 0 9 M -1, and 14,000 s i t e s / c e l l (Data not shown; in p r e p a r a t i o n ) . Thus, the differences between N B - M ~ and A d - M ~ in number and affinity of I F N - y receptors are apparently not substantial and within the range of technical error.

I/1

@

o o

E o. 10],]0 o

I

Z Lu ~3

z

5

O rn B (p moles)

0

I

I

1

2

APPLIED I F N - ~' ( n M ) FIG. 2

Quantitation of IFN-y specific M@ were incubated at 20 ° C for Specific binding was defined as insert represents the Scatchard and F, free IFN-~

binding to NB-M~. 2.5xi05 TGC-elicited NB4 hr with different amounts of 1 2 5 I - r I F N ~ described in M a t e r i a l s a n d M e t h o d s . The p l o t a n a l y s i s of the data. B, bound IFN-y

PGs are not involved in suppression of Ia-expression in NB-M~ We tested the possibility that a higher amount of PGs are produced in N B - M ~ cultures than in A d - M ~ cultures, and investigated the effect of IM , a PG s y n t h e s i s i n h i b i t o r (Fig. IA and Fig. IB). The p e r c e n t a g e of Ia +

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TABLE

I

Effect of preculture

IMain preculture b

--

--

IFN-

--+ + + + a. b. c. d. e.

with

IM

Reagents in culture b

--

y

c

IM d I F N - y + IM -IFN-y IM I F N - y + IM

649

% Ia + M@ Ad

NB

0.4

0.3

72.6

5.4

N.D. e 74.4 0.3 72.0 N.D. 69.4

N.D. 6.5 0.5 8.5 N.D. 7.6

1 ~ g / m l indomethacin For 3 days 30 IU/ml 1 pg/ml Not determined

c e l l s in IFN- y - s t i m u l a t e d A d - M ~ reached maximum at day 4 of the culture a n d t e n d e d to d e c l i n e t h e r e a f t e r in the a b s e n c e of IM, b u t the m a x i m u m r e m a i n e d in the p r e s e n c e of IM, s u g g e s t i n g t h a t e n d o g e n e o u s PGs downregulate Ia-expression of A d - M @ . No i m p r o v e m e n t by IM, h o w e v e r , was o b s e r v e d in Ia-expression of NB-M~. The ineffectiveness of IM for N B - M ~ might be due to the residual effect of PGs synthesized before the culture with I F N - y . In order to minimize this possibility, N B - M ~ were p r e c u l t u r e d for 3 days with d a i l y refreshment of the culture medium containing IM. The preculture of NB-M~, however, was ineffective in i n d u c i n g t h e i r r e s p o n s i v e n e s s to I F N - y (Table I), and s i m i l a r l y e v e n after p r e c u l t u r e of up to 5 days (data not shown). The p r e c u l t u r e of A d - M ~ did not a l t e r their r e s p o n s i v e n e s s to I F N - y I F N - ~ , but not I F N - ~ ,interferes with the response of NB-M~ to IFN-y It is still uncertain, however, whether I F N - ~ p l a y s a similar role to I F N - y in s u p p r e s s i n g Ia-expression on N B - M @ (4). To answer this question, M~ were c u l t u r e d with I F N - y in the presence of either antiI F N - ~ m A b or a n t i - I F N - ~ mAb. In c o n t r a s t to a n t i - I F N - ~ , anti-IFN-d did not improve r e s p o n s i v e n e s s of N B - M ~ (Table II). A n t i - I F N - d / 8 antibody showed a similar effect to anti-IFN- ~ (see Tables III,IV). These results indicate that N B - M ~ have an intrinsic potential to respond to I F N - y , but it is s u p p r e s s e d by IFN- ~ d e r i v e d f r o m some c e l l s in the N B - M @ preparation. I n d e e d , 1 0 - 5 0 I U / m l of I F N - ~ w a s d e t e c t a b l e in the N B - M @ culture supernatant, but IFN- ~ was u n d e t e c t a b l e (data not shown). Difference in the cell components between NB-M~ and Ad-M~ preparations. We u s e d 2 h r - a d h e r e n t T G C - P E C as a M ~ s o u r c e , of w h i c h the g r e a t majority were identifiable as M ~ by latex ingestion test. However, n o n - M ~ cells , mainly fibroblasts, w e r e a l s o f o u n d in the M @ p r e p a r a t i o n s especially in N B - M ~ We i n v e s t i g a t e d the c e l l components of M ~ preparation by the i n d i r e c t i m m u n o f l u o r e s c e n c e using F4/80 (anti-M~

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TABLE

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II

Effect of anti-IFN- ~

a

and anti-IFN-~ % Ia + M ~

3d Ad

5d NB

Ad

7d NB

Ad

NB

IFN- T plus --

74.0

3.9

82.5

7.3

63.7

1.8

Anti-IFN0.1~g/ml 1 ~g/ml

72.0 62.9

3.9 3.8

79.6 57.0

5.1 3.7

61.5 46.9

3.1 0.9

Anti-IFN0.01~g/ml 0.i ~ g / m l 1 ~g/ml

72.2 72.1 74.5

23.1 26.6 10.6

80.0 76.1 69.6

31.1 30.3 32.3

58.9 58.9 59.4

21.1 17.1 27.5

a. M~ were cultured for 3-7 days with IFN- T (30U/ml) and anti-IFN- ~ mAb or anti-IFN-~ mAb.

a n t i g e n ) , 2.4G2 (anti-FcR), H O - 1 3 - 4 (anti-Thy-l.2) and a l s o 10.2.16 (antiIa k) (Table III). Percentage of F4/80 + c e l l s and that of 2.4G2 + c e l l s were independent of the emergence of Ia + c e l l s in the culture. Similar trends were a l s o o b s e r v e d in N B - M ~ preparation, though the percentages of F4/80 + and 2.4G2 + c e l l s w e r e s l i g h t l y l o w in c o m p a r i s o n w i t h A d - M @ . In A d - M ~ p r e p a r a t i o n , v i r t u a l l y no T h y - i + c e l l was d e t e c t e d . On the o t h e r hand, low but substantial percentages of Thy-i + c e l l s (3-10%) were found in NBM@ preparation, and t h e y a p p e a r e d to be f i b r o b l a s t s , not T c e l l s , morphologically. Increase in the percentage of Thy-i + c e l l s in the N B - M @

TABLE

III

Cell components in Ad-M~ and NB-M@ preparations % positive cells F4/80 2.4G2

Cultured with

(2d/4d cultured) Thy-I

Ia

Ad -Anti-IFN-~/~ IFN-T b IFN-T + Anti-IFN-~/~

98.6/99.6 98.6/98.9 98.4/95.8 98.1/98.2

96.3/97.4 97.7/96.3 97.0/97.2 96.9/97.6

<0.i/<0.i <0.i/<0.I <0.i/<0.i <0.i/<0.i

<0.1/3.1 <0.1/3.1 75.2/83.9 74.3/82.0

96.9/92.5 96.9/92.5 96.9/95.7 95.3/95..1

93.1/92.8 93.1/92.8 94.5/93.2 95.2/92.8

3.2/5.5 3.2/5.4 2.4/6.9 3.3/5.9

<0.i/<0.I <0.i/<0.i 2.7/4.7 11.3/22.1

NB

-Anti-IFN-~/~ IFN-y IFN- T + Anti-IFN- ~ / ~ a. Mona 2 A b. 20 U/ml

, 20~ul/ml

(i ~ul neutralize i0 U I F N - e / ~

)

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3

la EXPRESSION OF NEONATAL MACROPHAGES

culture (Table III,IV) increase of fibroblast

was due to the decrease number.

TABLE

Effect of depletion

of

Culture with IFN-T b IFN- T + Anti-IFN-~/~ -

AntiThy-i

IFN- y IFN-T + Anti- IFN- ~ / ~ -

C'

IFN-y IFN-T + Anti- IFN- ~ / 8

AntiThy-i + C'

a. b. c. d.

-

IFN-y IFN-~ + Anti-IFN- ~ / ~

d 20.8 i.i 5.3

% Thy-i + 5d

(2.4) a (98.9) (98.9)

(1.8) (97.1)

24.6

(99.4)

0.7 5.0

cells

3d

1.2 3.8

N.D.

20.1

but not the

of Thy-i + fibroblasts

3d 0.5 4.2

M ~ number,

IV

% Ia + M@ Pretreatment

651

5d

3.3 (<0.i) N.D. c N.D.

N.D.

8.3 8.3

(<0.i)

10.5 N.D.

20.1

1.0

(4.6)

1.4

(1.4)

4.1

(98.1)

6.6

(97.1)

3.8

N.D.

N.D.

20.1

(96.5)

20.1

(97.3)

N.D.

N.D.

(4.4)

0.9

(1.9)

(98.8)

4.4

(96.9)

N.D.

0.9

(N.D.)

21.3

(97.9)

19.8

(98.7)

N.D.

0.i

(N.D.)

the percentage

0.6

(N.D.)

1.9

indicate

(<0.i)

8.6

6.4

Figures in parentheses 20 U/ml Not determined 20 pl/ml

0.i

(N.D.)

(<0.i)

in Ad-M~.

Effect of depleting fibroblasts S i n c e f i b r o b l a s t s are k n o w n as a p r o d u c e r of I F N - 8 , c o n t a m i n a t i n g f i b r o b l a s t s might produce IFN- 8 in our N B - M ~ culture. We confirmed this p o s s i b i l i t y by treating N B - M ~ preparations with anti-Thy-i mAb plus C' in order to deplete fibroblasts before initiating cultures with IFN- T (Table IV). A n t i - T h y - i m A b p l u s C' t r e a t m e n t d e p l e t e d T h y - i + c e l l s e v e n in 3-5 d a y s c u l t u r e of N B - M ~ . O n l y v e r y f e w Ia + c e l l s emerged, however, in f i b r o b l a s t - d e p l e t e d N B - M ~ preparations as w e l l as u n d e p l e t e d preparations w h e n c u l t u r e d w i t h IFN- T a l o n e . A p p r e c i a b l e d e g r e e of I a - i n d u c t i o n by IFN- ~ was o b s e r v e d only when anti-IFN- d / ~ was further added. Thus, the contaminating fibroblasts were not r e s p o n s i b l e for inhibiting Ia-expression of NB-M~. Rather, N B - M ~ t h e m s e l v e s seem to produce IFN-8 which suppresses Ia-expression in N B - M ~ population.

Discussion

We reported

previously

that N B - M ~

did not e a s i l y

express

Ia

antigens

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even if stimulated with IFN-y. They did become responsive to I F N - y , though weaker than Ad-M@, by the n e u t r a l i z a t i o n of IFN- 8 in N B - M ~ culture (4). The present study was undertaken as an extension of the p r e v i o u s one to elucidate the f o l l o w i n g points; i) Is low responsiveness of N B - M ~ to I F N - y attributed, at least in part, to the inferiority of N B - M 0 to A d - M @ in the q u a n t i t y a n d / o r the q u a l i t y of I F N - 7 receptors? 2) A r e o t h e r factors such as PGs or IFN- ~ also i n v o l v e d in the suppression of response of N B - M ~ to IFN- y ? a n d 3) F r o m w h a t c e l l s is the s u p p r e s s i v e factor derived? As mentioned above, a part of N B - M ~ become responsive to I F N - y in the presence of a n t i - I F N - 8 under in v i t r o conditions. A l t h o u g h this indicated the presence of functional IFN- y receptors on some, if not all, of N B - M ~ among those treated with anti-IFN-~ , it w a s s t i l l u n c e r t a i n w h e t h e r e v e n untreated N B - M ~ expressed IFN-y receptors as w e l l as Ad-M~. As shown in Fig. 2, the u n t r e a t e d NB-M@ possess IFN-y receptors (25,000 sites/cell a n d K a = 3 . 8 x 1 0 9 M-l). This value is not s u b s t a n t i a l l y d i f f e r e n t f r o m t h a t of A d - M ~ (14,000 s i t e s / c e l 1 a n d K a = l . 7 x l 0 9 M -1) as described before. Similar v a l u e s in A d - M ~ have also been reported by C e l a d a et al. (i0,ii). This c l e a r l y indicates that the low r e s p o n s i v e n e s s of naive NB-M@ to IFN-y for Ia-expression is not due to the lack of IFN-y receptors. Inhibitory effect of PGs on Ia-expression of M~ was reported by Snyder et al. (5,7,13). They demonstrated also that newborn mouse spleens contained some suppresser cells, p o s s i b l y M~ precursors, which seemed to deliver PGs. We i n f e r r e d f r o m t h i s t h a t o u r N B - M ~ p o p u l a t i o n s might c o n t a i n the p r o d u c e r of s u c h s u p p r e s s i v e PGs. This s e e m e d i m p l a u s i b l e , however, since neither coculture of N B - M @ with IM and I F N - y nor preculture of them with IM before r e c e i v i n g IFN- ~ was ineffective in restoring their low r e s p o n s i v e n e s s to I F N - y (Fig. 1 and T a b l e I). As mentioned before, b o t h IFN- ~ a n d IFN- 8 are r e p o r t e d to be inhibitory for Ia-induction of A d - M ~ by I F N - y (4,14), and, as for N B - M ~ , we p r e s e n t e d in the p r e v i o u s paper o n l y the result that the response of N B - M ~ to IFN-7 was augmented by anti-IFN-~ ( 4 ) . Thus, the e f f e c t of a n t i - I F N - d w a s e x a m i n e d in the p r e s e n t study. Results indicate that IFN-~ is not i n v o l v e d in the apparent low r e s p o n s i v e n e s s to I F N - y of c u l t u r e d NB-M~. Our N B - M @ preparations contained low but substantial percentages of f i b r o b l a s t s b e a r i n g T h y - i a n t i g e n s (15,16) ( T a b l e III). F i b r o b l a s t s are k n o w n to be a p o t e n t p r o d u c e r of I F N - ~ (17). The p o s s i b i l i t y t h a t I F N - ~ , produced by f i b r o b l a s t s , suppresses Ia-expression of N B - M ~ seems implausible, since NB-M~ d e p r i v e d of f i b r o b l a s t s are s t i l l m i n i m a l l y r e s p o n s i v e to I F N - y ( T a b l e IV). Thus, it is m o s t p r o b a b l e t h a t N B - M ~ themselves produce IFN-~ to d o w n - r e g u l a t e their Ia-expression. This is c o n s i s t e n t w i t h the s u p p r e s s i v e e f f e c t of e x o g e n e o u s IFN- ~ on the Iaexpression of Ad-M~(4,14), and also w i t h t h a t of i n t r a p e r i t o n e a l administration of LPS or p o l y I:C, w h i c h are k n o w n as p o t e n t IFN- ~ inducers, on I a - e x p r e s s i o n of A d - M @ under in v i v o condition (our u n p u b l i s h e d data). Thus, the questions described in the beginning of this section were answered, and a considerably clear conclusion has b e e n obtained : The low r e s p o n s i v e n e s s of N B - M ~ to I F N - ~ is m a i n l y due to the suppressive effect of IFN- ~ d e r i v e d from N B - M ~ themselves, though it is u n c e r t a i n w h e t h e r the r e s p o n d e r M ~ a n d the s u p p r e s s i v e M ~ are d i f f e r e n t from or identical with each other. Other

possibilities,

however,

also

remain

whether

anti-IFN-~

may

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653

a c c e l e r a t e the maturation of N B - M @ to the adult type, and that the antibody may injure preferentially some M~ which exert suppressive effects independently of IFN-~ secretion. We obtained no evidence for or against these p o s s i b i l i t i e s in the present investigation only by monitoring surface markers and v i a b i ] i t i e s of N B - M ~ preparation during the culture for 4 days (Table III). The percentage of responsive N B - M ~ to IFN- T d i s c l o s e d by a n t i - I F N - 8 was still lower than that of responsive Ad-M@. This may be simply due to the small p o p u l a t i o n size of the intrinsic responder M~ in NB-M~. Other possibilities are t h a t a n t i - I F N - ~ in the c u l t u r e m e d i u m is a b l e to neutralize only e x t r a c e l l u l a r IFN- 8 but not i n t r a c e l l u l a r IFN-8 , and that a l s o some factors other than IFN- ~ participate in the suppression of I a - e x p r e s s i o n of N B - M @ . It was r e p o r t e d t h a t c r u d e C S F s i n h i b i t e d Iaexpression of M~ (18,19), and we a l s o o b s e r v e d the suppressive effects of crude CSF-I, crude IL-3 (WEHI-3 culture supernatant), or purified G M - C S F on Ia-expression of A d - M @ (our u n p u b l i s h e d data). In addition, CSFs stimulate M~ to p r o l i f e r a t e and to produce I F N - ~ which in turn down-regulates the p r o l i f e r a t i o n of M ~ (8,20). It r e m a i n s to be i n v e s t i g a t e d , t h e r e f o r e , whether CSFs h a v e some connection with low Ia-expression of NB-M~. It is we] i k n o w n t h a t the e x p r e s s i o n of c l a s s I and c l a s s II m a j o r h i s t o c o m p a t i b i l i t y (MHC) antigens is r e g u l a t e d by IFNs not o n l y in adult but a l s o in f e t a l and n e o n a t a l a n i m a l s (21,22). O u r p r e s e n t r e s u l t s may present a t e l e o l o g i c a l l y reasonable conception for d e v e l o p m e n t a l biology. I F N - 8 p r o m o t e s the e x p r e s s i o n of c l a s s I M H C a n t i g e n s as g e n u i n e s e l f m a r k e r s but s u p p r e s s e s t h a t of c l a s s II M H C a n t i g e n s , w h i c h s e r v e as immune-initiating signals. This may a v o i d premature d e v e l o p m e n t of immune responsiveness thus establishing self-tolerance. The findings of Lu et al., that I a - b e a r i n g a c c e s s o r y c e l l s w h i c h t r i g g e r T l y m p h o c y t e s f o u n d e a r l y in the t h y m u s and l a t e r in the s p l e e n d u r i n g o n t o g e n y , m a y s u p p o r t this c o n c e p t (23).

Acknowledgments We thank all members of our laboratory for technical assistance and discussions. T h i s w o r k was s u p p o r t e d by a G r a n t - i n - A i d for S c i e n t i f i c R e s e a r c h f r o m the M i n i s t r y of E d u c a t i o n , S c i e n c e , and C u l t u r e of Japan, and the Shimizu Foundation Research Grant.

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