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Attachment and phagocytosis by salmon macrophages of agarose beads coated with human C3b and C3bi
DEVELOPMENTAL ~ND COMPARATIVE IMMUNOLOGY, Vol. 8, pp. 623-630, 1984. 0145-305X/84 $3.00 + .00 Printed in the USA. Copyright (c) 1984 Pergamon Press Ltd. All rights reserved.
A T T A C H M E N T AND P H A G O C Y T O S I S
BY SALMON M A C R O P H A G E S
OF A G A R O S E BEADS C O A T E D WITH HUMAN C3b and C3bi Egil J o h n s o n and P a t r i c k Smith Institute of M e d i c a l Biology, D e p a r t m e n t of M o r p h o l o g y I, U n i v e r s i t y of Troms@, N - 9 0 0 1 Troms~, N o r w a y
ABSTRACT
A g a r o s e beads (diameter 5-10 ~m) p r e i n c u b a t e d in h u m a n serum b e c a m e a s s o c i a t e d (attached and ingested) to 50-60% of the salmon m a c r o p h a g e s w i t h i n 60 m i n u t e s However, Beads p r e i n c u b a t e d in serum t r e a t e d w i t h h e a t i n g (50°C, 20 min) or w i t h EDTA (i0 mM) to inhibit the a c t i v a t i o n of a l t e r n a t i v e c o m p l e m e n t pathway, were not a s s o c i a t e d to the p h a g o c y t e s . F u r t h e r m o r e , a g a r o s e b e a d s c o a t e d w i t h h u m a n C3b and C3bi after i n c u b a t i o n w i t h i s o l a t e d c o m p l e m e n t factors (C3, D, B), were a s s o c i a t e d to 30-40% of the phagocytes. A b o u t 80% of the c e l l - a s s o c i a t e d a g a r o s e beads was i n t r a c e l l u l a r l y located. C o n v e r s i o n by t r y p s i n t r e a t m e n t (0.01%) of a g a r o s e b o u n d C3bi to C3d, a b o l i s h e d the a s s o c i a t i o n of such beads to the m a c r o p h a g e s . The results d e m o n s t r a t e that salmon m a c r o p h a g e s p o s e s s c o m p l e m e n t r e c e p t o r s that bind h u m a n C3b and C3bi. A g a r o s e beads c o a t e d with these ligands (C3b and C3bi) are a t t a c h e d and i n g e s t e d by the phagocytes.
INTRODUCTION We have p r e v i o u s l y shown that the g l y c a n (polysaccharide) agarose s t i m u l a t e m o u s e m a c r o p h a g e s in v i t r o as m e a s u r e d by i n c r e a s e d 1 ~ C - g l u c o s a m i n e i n c o r p o r a t i o n into m a c r o m o l e c u l e s 623
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r e d u c e d 5 " - n u c l e o t i d a s e activity, i n c r e a s e d cell spreading (13,18) and c y t o t o x i c e f f e c t on tumor cells (9). We looked into the m e c h a n i s m b e h i n d the s t i m u l a t o r y e f f e c t of agarose on the m a c r o p h a g e s . Our main finding was that agarose must be e n d o c y t o s e d by the m a c r o p h a g e s in order to exert its stimulatory e f f e c t (9). A g a r o s e like other s t i m u l a t o r y glycans, are p o t e n t a c t i v a t o r s of the a l t e r n a t i v e p a t h w a y of c o m p l e m e n t (13,18). We found that agarose must be c o a t e d with C3b or C3bi before a t t a c h m e n t and u p t a k e of the g l y c a n by the c o r r e s p o n d i n g r e c e p t o r s for C3b and C3bi on both m o u s e m a c r o p h a g e s (8,11,12) and human m o n o c y t e s (8,10). F r o m this it was clear that c o m p l e m e n t c o a t e d agarose beads d i a m e t e r (5-10 ~m) was a novel and e x c e l l e n t p a r t i c l e for s t u d y i n g the o c c u r e n c e of c o m p l e m e n t r e c e p t o r s on m o n o n u clear phagocytes. The c o m p l e m e n t s y s t e m and c o m p l e m e n t receptors on m o n o n u c l e a r p h a g o c y t e s are e s s e n t i a l for the p r o t e c t i o n of the host a g a i n s t m i c r o o r g a n i s m s . T h e r e f o r e , it is of biological i n t e r e s t to i n v e s t i g a t e w h e t h e r p h a g o c y t e s further down in the animal k i n g d o m p o s e s s receptor(s) for c o m p l e m e n t factor C3, and w h e t h e r human c o m p l e m e n t p o s s i b l y can interact with such receptors. We found that salmon m a c r o p h a g e s p o s e s s r e c e p t o r s for h u m a n C3b and C3bi. A g a r o s e beads c o a t e d with these ligands are a t t a c h e d and i n g e s t e d by the p h a g o c y t e s .
_MATERIALS AND M E T H O D S E x p e r i m e n t a l fish. Salmon (10-20 g) from fresh w a t e r was o b t a i n e d from J . E . P e t t e r s e n , B r e e d i n g Station, K v a l ~ y v~gen, Norway, and kept in tanks of d e c h l o r i n a t e d water at 14-16oc until use. While in the l a b o r a t o r y the fish were fed w i t h Tess salmon food. C u l t u r e medium. The m e d i u m used for p r e p a r a t i o n of cell s u s p e n s i o n and m a i n t e n a n c e in c u l t u r e was L-15 (Leibovitz, G r a n d Island, B i o l o g i c a l Company, U.S.A.) s u p p l e m e n t e d with 0.033 g g l u c o s e / m l , 100 IU p e n i c i l l i n - s t r e p t o m y c i n / m l (Gibco Biocult, Scotland) and 10% heat i n a c t i v a t e d (56°C, 30 min) foetal calf serum (FCS) (Gibco). M e d i u m was a d j u s t e d to pH 7.6 and m O s m of 320. P r e p a r a t i o n of l y m p h o i d cells. The salmon m a c r o p h a g e s were i s o l a t e d largely as p r e v i o u s l y d e s c r i b e d (5). However, there are m i n o r m o d i f i c a t i o n s r e g a r d i n g the d e n s i t i e s of the p e r c o l l g r a d i e n t s used, and the m e t h o d will be d e s c r i b e d in detail. Fish were k i l l e d by d e c a p i t a t i o n . The p r o n e p h r o s was d i s s e c t e d out by v e n t r a l incision, c a r e f u l l y lifted out w i t h c u r v e d forceps and t r a n s f e r r e d to 3 ml L-15 medium. The cell s u s p e n s i o n was p r e p a r e d by t e a s i n g the p r o n e p h r o s with two bent glass rods over a s t a i n l e s s steel m e s h (diameter 0.03 mm) in a small glass dish. Clumps of cells were r e m o v e d after s e d i m e n t a t i o n in a test tube (Falcon, C o c k e y s v i l l e , U.S.A.),
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and the cells r e m a i n i n g in s u s p e n s i o n were c o u n t e d and their v i a b i l i t y t e s t e d by t r y p a n blue e x c l u s i o n (0.05% solution). D i f f e r e n t i a l c e n t r i f u ~ a t i o n of l y m p h o i d cells. The cell s u s p e n s i o n was loaded onto Percoll g r a d i e n t s (Pharmacia Fine Chemicals, Uppsala, Sweden) in 25 ml tubes (Nunco) for d e n s i t y centrifugation. Percoll was d i l u t e d in L-15 to give the diffe r e n t g r a d i e n t d e n s i t i e s of 1.060, 1.070 and 1.090, respectively. The g r a d i e n t s w e r e c a r e f u l l y l a y e r e d from the b o t t o m of the tubes as follows: 1.090 (2.5 ml), 1.070 (5.0 ml) and 1.060 (2.5 ml). Then the cell s u s p e n s i o n in L-15 (3.0 ml) was g e n t l y l a y e r e d on top of the g r a d i e n t s and the tube was c e n t r i fuged at 400 g in a D a m o n / I E C D i v i s i o n c e n t r i f u g e Pr-6000. The cell b a n d c o n t a i n i n g 80-90% m a c r o p h a g e like cells plus some l y m p h o c y t e s and p o l y m o r p h o n u c l e a r g r a n u l o c y t e s , was i d e n t i f i e d b e t w e e n the upper two g r a d i e n t s in the tube. In v i t r o c u l t u r e conditions. The cell s u s p e n s i o n from the u p p e r cell b a n d was w a s h e d in L-15 m e d i u m s u p p l e m e n t e d w i t h FCS and counted. A b o u t 0.8 x 106 c e l l s / m l were seeded on glass c o v e r s l i p s (diameter 14 mm) in C O s t a r tissue c u l t u r e p l a t e s (Costar, C a m b r i d g e , Mass., U.S.A.). The cells were c u l t u r e d at 10°C in air and w a s h e d three times in L-15 after 2 hours of i n c u b a t i o n to r e m o v e n o n - a d h e r e n t cells, and f u r t h e r c u l t u r e d in L-15 w i t h FCS. All e x p e r i m e n t s were p e r f o r m e d after 3 days of i n c u b a t i o n u n d e r s e r u m f r e e conditions, after w a s h i n g to r e m o v e FCS. In 3 days old c u l t u r e s about 90-98% of the cells w e r e well s p r e a d and c a p a b l e of p h a g o c y t o s i s (fiwed sheep e r y t h r o c y t e s , h e a t k i l l e d C a n d i d a and living Yeast cells) (5), w h i c h are c r i t e r i a for the i d e n t i f i c a t i o n of m a c r o p h a g e s . H u m a n serum. Human b l o o d was o b t a i n e d by v e n i p u n c t u r e of h e a l t h y young donors and a b s o r b e d by p a s s a g e t h r o u g h a S e p h a r o s e 4B (Pharmacia) c o l u m n at 4°C to r e m o v e C - r e a c t i v e p r o t e i n (ii) and any a n t i b o d i e s a g a i n s t a g a r o s e (8). P r e i n c u b a t i o n of a ~ a r o s e beads in h u m a n serum. Agarose beads (diameter 5-10 Hm, 1 mg = 6 x 106 beads) was a gift from P h a r m a c i a Fine Chemicals, Uppsala, Sweden. The a g a r o s e beads at a c o n c e n t r a t i o n of 1 mg/ml were i n c u b a t e d in h u m a n serum d i l u t e d 1:2 for 15 m i n u t e s at 37oc, and then w a s h e d three times in L-15 by c e n t r i f u g a t i o n at 450 g for 5 minutes. The beads w e r e n o w m a i n l y c o a t e d w i t h C3bi (8,11,15). P r e p a r a t i o n of a @ a r o s e beads c o a t e d w i t h C3b and C3bi. The m e t h o d of c o a t i n g a g a r o s e beads w i t h h u m a n C3b and C3bi by i n c u b a t i o n w i t h t r y p s i n and f u n c t i o n a l l y pure c o m p l e m e n t f a c t o r s of the a l t e r n a t i v e p a h t w a y (C3, B, D) has been d e s c r i bed (8,11). The i n c u b a t i o n of the beads w i t h B, D and C3 was r e p e a t e d five times. By also using ~ 2 S I - l a b e l l e d (6) C3 to a s p e c i f i c a c t i v i t y of 0.3 H C i / ~ , the a m o u n t of 12sI on 1 ~g a g a r o s e beads was a b o u t 5 x i0 J c.p.m, g i v i n g a b o u t 1.7 Hg p r o t e i n per 100 ~g a g a r o s e (ii). One half of the C3b c o a t e d a g a r o s e beads was t r e a t e d with h u m a n serum d i l u t e d 1:20 in g e l a t i n e - b a r b i t a l b u f f e r (GVB) c o n t a i n i n g i0 mM e t h y l e n e d i a minetetraacetate (EDTA) for 15 m i n u t e s to c o n v e r t a g a r o s e
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l i n k e d C3b to C3bi. A b o u t 83% of a g a r o s e b o u n d C,3b w a s c o n v e r ted to C3bi, as p r e v i o u s l y s h o w n w i t h the use of rat m o n o c l o n a l a n t i - h u m a n C3c and - C 3 d a n t i b o d i e s (8,14). A s s o c i a t i o n of a ~ a r o s e b e a d s to the m a c r o p h a ~ e s . Beads w e r e a d d e d to t h e p h a g o c y t e s at a b e a d to cell r a t i o of 3:1. A f t e r i n c u b a t i o n for 60 m i n u t e s at 10°C the c e l l s w e r e w a s h e d c a r e f u l l y t h r e e t i m e s by d i p p i n g the c o v e r s l i p s in w a r m L-15 to r e m o v e free a g a r o s e b e a d s , a n d t h e n f i x e d in 2.5% g l u t a r a l d e h y d e , 0.i M s u c r o s e in 0.i M c a c o d y l a t e b u f f e r , pH 7.3. Association ( a t t a c h m e n t and i n g e s t i o n ) of a g a r o s e b e a d s to the p h a g o c y t e s was e x a m i n e d in a p h a s e c o n t r a s t m i c r o s c o p e (Carl Zeiss) and d e f i n e d as the p e r c e n t a g e of the t o t a l cell p o p u l a t i o n a s s o c i a t e d w i t h at l e a s t one a g a r o s e bead. The dist i n c t i o n b e t w e e n a t t a c h m e n t and i n g e s t i o n was e v a l u a t e d by o b s e r v i n g m i c r o s c o p i c a l l y w h e t h e r the o u t e r c e l l m e m b r a n e e n c l o s e d the e n t i r e a g a r o s e bead. Micro~raphs. The macrophage cultures were fixed m i c r o g r a p h s w e r e t a k e n in a p h a s e c o n t r a s t m i c r o s c o p e .
before
RESULTS Cell-association of a @ a r o s e b e a d s p r e i n c u b a t e d in h u m a n s e r u m or c o a t e d w i t h C 3 b or C3bi. S i n c e a g a r o s e is an a c t i v a t o r of the a l t e r n a t i v e p a t h w a y of c o m p e l e m e n t , i n c u b a t i o n in s e r u m i n i t i a l l y g i v e s a g a r o s e l i n k e d C3b. H o w e v e r , a f t e r 15 m i n u t e s of i n c u b a t i o n the b e a d s a r e m a i n l y c o a t e d w i t h C 3 b i (ii,12,15). S u c h b e a d s w e r e a d d e d to the m a c r o p h a g e s for 60 m i n u t e s , and 5 0 - 6 0 % of the t o t a l c e l l p o p u l a t i o n w a s a s s o c i a ted to the a g a r o s e b e a d s (data n o t s h o w n ) . U s u a l l y the p h a g o c y t e s w e r e a s s o c i a t e d to o n e b e a d , o c c a s i o n a l l y two b e a d s . A g a r o s e b e a d s p r e i n c u b a t e d in h u m a n s e r u m t r e a t e d w i t h i0 ~ 4 E D T A or w i t h h e a t i n g (50oc, 20 min) as w e l l as n a t i v e a g a r o s e b e a d s , w e r e n o t a s s o c i a t e d to the m a c r o p h a g e s (Table I) . TABLE Association
of
serum
I
treated
T r e a t m e n t of aHarose beads
a@arose
beads
to m a c r o p h a g e s
x Association
Human serum (15 min, 37°C) Human serum (15 min, 37oc) E D T A (I0 mM) 50°C, 20 m i n None x
+ = Association (attachment - = no a s s o c i a t i o n
and
ingestion),
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A b o u t 30-40% of the total cell p o p u l a t i o n w a s a s s o c i a t e d to the b e a d s c o a t e d w i t h C3b or C3bi (Figures 1 a) and 1 b)). As e v a l u a t e d by p h a s e c o n t r a s t m i c r o s c o p y , a b o u t 80% of the a g a r o s e ( p r e i n c u b a t e d in serum, c o a t e d w i t h C3b or C3bi) was i n t r a c e l l u l a r l y l o c a t e d (Figures 1 a) and 1 b)), w h i c h is in a c c o r d a n c e w i t h c o m p a r a b l e s t u d i e s on h u m a n m o n o c y t e s (I0) and m o u s e m a c r o p h a g e s (ii). The p h a g o c y t e s b e c a m e less s p r e a d and p a r t l y d e t a c h e d f r o m the g l a s s c o v e r s l i p s , a f t e r i n g e s t i o n of the a g a r o s e b e a d s (Figures 1 a) a n d 1 b)). T r y p s i n t r e a t m e n t (0.01% for 30 m i n u t e s a t 37°C) of C3bi c o a t e d a g a r o s e beads to c o n v e r t C3bi to m a i n l y C 3 d (8), abolished the a s s o c i a t i o n of such b e a d s to the p h a g o c y t e s (Table II). However, t r y p s i n i z a t i o n of a g a r o s e l i n k e d C3b did not r e d u c e a s s o c i a t i o n of these b e a d s to the m a c r o p h a g e s , since C3b is r e l a t i v e l y r e s i s t a n t to short e x p o s u r e s of t r y p s i n u n d e r these c o n d i t i o n s (8). TABLE Association
of c o m p l e m e n t
Agarose beads coated with C3b C3b C3bi C3bi
coated
II a~arose
Treatment None Trypsin None Trypsin
(0.01%) (0.01%)
a)
beads
to m a c r o p h a g e s
Association + + + -
b) FIG
I
Phase c o n t r a s t m i c r o g r a p h s (x 400) of s a l m o n m a c r o p h a g e s . A g a r o s e beads c o a t e d w i t h C 3 b (a) and C3bi (b) are a s s o c i a t e d to m a n y of the p h a g o c y t e s . The beads are i n d i c a t e d by b l a c k arrows.
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DISCUSSION We p r e s e n t e v i d e n c e that salmon m a c r o p h a g e s p o s e s s comp l e m e n t r e c e p t o r s that bind h u m a n c o m p l e m e n t factors C3b and C3bi. F u r t h e r m o r e , that c o m p l e m e n t (C3b or C3bi) c o a t e d a g a r o s e beads are p h a g o c y t o s e d by the phagocytes. A s s o c i a t i o n of serum t r e a t e d beads was d e p e n d e n t on an i n t a c t f u n c t i o n of the a l t e r n a t i v e c o m p l e m e n t p a h t w a y (Table I), since i n a c t i v a t i o n of factor B (50°C, 20 minutes) or p r e s e n c e of E D T A (!0 ~M) a b o l i s h e d the a s s o c i a t i o n of the beads to the cells. This m e a n s that a c t i v a t i o n of the a l t e r n a t i v e p a t h w a y of c o m p l e m e n t is a p r e r e q u i s i t e for the a s s o c i a t i o n of the a g a r o s e to the m a c r o p h a g e s . The i n c r e a s e d a s s o c i a t i o n (20-30%) to the p h a g o c y t e s of serum t r e a t e d beads c o m p a r e d to C3b and C3bi c o a t e d beads, is p r o b a b l y m a i n l y due to more c o m p l e m e n t (mainly C3bi) (8,11,15) on the former beads. However, this d i f f e r e n c e may also be r e l a t e d to b i n d i n g of a d d i t i o n a l serum p r o t e i n s to the beads, e s p e c i a l l y C5b (ii) and f i b r o n e c t i n w h i c h i n t e r a c t s with factor C3 (7). C3b and C3bi c o a t e d agarose b e a d s a s s o c i a t e d e q u a l l y to the p h a g o c y t e s , even though C3bi has h i g h e r a f f i n i t y for the C3bi r e c e p t o r than C3b for the C3b r e c e p t o r (8,10,19). Thus our finding is m o s t likely c a u s e d by the large a m o u n t of agarose linked c o m p l e m e n t (1.7 ~g p r o t e i n per i00 ~g agarose), w h i c h d i m i n i s h the s i g n i f i c a n e of p r o s p e c t i v e d i f f e r e n c e s in a f f i n i t i e s b e t w e e n the ligands and their c o r r e s p o n d i n g receptors. A n o t h e r p o s s i b i l i t y is that the salmon m a c r o p h a g e s only p o s e s s a single c o m p l e m e n t C3 r e c e p t o r that can not d i s t i n q g u i s h b e t w e e n human C3b and C3bi due to the species incompatibility. However, this e x p l a n a t i o n is less likely c o n s i d e r i n g the separate r e c e p t o r s for C3b and C3bi r e p o r t e d (1,8,10,17,19) on b o t h m o u s e m a c r o p h a g e s and h u m a n m o n o c y t e s . T r y p s i n treatm e n t of the C3bi c o a t e d a g a r o s e beads (Table II), w h i c h r e m o v e d a b o u t 83% of a g a r o s e b o u n d C3c (8), a b o l i s h e d the a s s o c i a t i o n of such beads (mainly C3d coated) to the p h a g o c y t e s , w h e r e a s the t r y p s i n r e s i s t a n t C3b c o a t e d beads still a s s o c i a t e d to the cells. These r e s u l t s c l e a r l y d e m o n s t r a t e that the a t t a c h m e n t of c o m p l e m e n t c o a t e d a g a r o s e to the C3b and C3bi receptors, is m e d i a t e d via the C3c p o r t i o n of the C3 molecule. The k i n e t i c s of the i n g e s t i o n of agarose by the phagocytes was not s t u d i e d in detail. However, we c o n c l u d e that the u p t a k e r e a c h e d a p l a t e a u w i t h i n one hour, since longer incubation time did not increase the a m o u n t of beads a s s o c i a t e d to the cells (data not shown). The general c o n c e p t is (4,16,19) that r e s i d e n t m o n o n u clear p h a g o c y t e s (mouse m a c r o p h a g e s , human monocytes) only bind C3b and C3bi c o a t e d sheep e r y t h r o c y t e s , w h i c h are not i n g e s t e d u n l e s s the p h a g o c y t e s are s t i m u l a t e d with specifig agents. On the contrary, this study and p r e v i o u s results (8,10,11) have shown that n o n o n u c ! e a r p h a g o c y t e s ingest comp l e m e n t c o a t e d a g a r o s e beads i n d e p e n d e n t of s p e c i f i c stimulation. This d i s c r e p a n c y in r e s u l t s m u s t be r e l a t e d to the d i f f e r e n t n a t u r e of the test p a r t i c l e s being used (agarose
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beads v e r s u s sheep e r y t h r o c y t e s ) . F u r t h e r m o r e , we found (10, ii) a c o r r e l a t i o n b e t w e e n i n g e s t i o n of test p a r t i c l e s by the p h a g o c y t e s and their a b i l i t y to a c t i v a t e the a l t e r n a t i v e pathway of complement. R e c e n t l y it is also r e p o r t e d that e c h i n o i d p h a g o c y t e s (2, 3) bind and i n g e s t sheep e r y t h r o c y t e s c o a t e d with human C3bi. The wide d i s t r i b u t i o n of c o m p l e m e n t like r e c e p t o r s in mammals, fish and i n v e r t e b r a t e s , e m p h a s i z e the s i g n i f i c a n c e of complem e n t as a host s y s t e m a g a i n s t m i c r o o r g a n i s m s .
ACKNOWLEDGEMENTS This work was s u p p o r t e d by g r a n t s from The N o r w e g i a n R e s e a r c h C o u n c i l for S c i e n c e and the H u m a n i t i e s and the N o r w e g i a n F i s h e r i e s R e s e a r c h Council.
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B E R T H E U S S E N , K. and SELJELID, R. R e c e p t o r s for c o m p l e m e n t on e c h i n o i d p h a g o c y t e s . I. The o p s o n i c effect of v e r t e b r a t e sera on e c h i n o i d p h a g o c y t o s i s . Dev. Comp. Immunol. 6, 423, 1982.
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B E R T H E U S S E N , K. R e c e p t o r s for c o m p l e m e n t on e c h i n o i d p h a g o cytes. II. P u r i f i e d h u m a n c o m p l e m e n t m e d i a t e s e c h i n o i d phagocytosis. Dev. Comp. Immunol. 6, 635, 1982. BIANCO, C., GRIFFIN, F.M. and S I L V E R S T E I N , S.C. Studies on the m a c r o p h a g e c o m p l e m e n t receptors. A l t e r a t i o n of receptor f u n c t i o n upon m a c r o p h a g e a c t i v a t i o n . J. exp. Med. 141, 1278, 1975.
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B R A U N - N E S J E , R., B E R T H E U S S E N , K., KAPLAN, G. and SELJELID, R. S a l m o n i d m a c r o p h a g e s : separation, in vitro culture and characterization. J. Fish D i s e a s e s 4, 141, 1981.
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FRAKER, P.J. and SPECK, J.C. P r o t e i n and cell m e m b r a n e i o d i n a t i o n s w i t h a s p a r i n g l y soluble c h l o r o a m i d e , 1,3,4,6,tetra-chloro-3a,6a-diphenyl glucoluril. Biochem. Biophys. Res. Commun. 80, 849, 1978.
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HAUTANEN, A. and K E S K I - O J U , J. I n t e r a c t i o n of f i b r o n e c t i n w i t h c o m p l e m e n t c o m p o n e n t C3. Scand. J. Immunol. 17, 225, 1983.
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JOHNSON, E., B@GWALD, J., ESKELAND, T. and SELJELID, R. C o m p l e m e n t ( C 3 ) - r e c e p t o r m e d i a t e d a t t a c h e m e n t of a g a r o s e beads to m o u s e p e r i t o n e a l m a c r o p a h g e s and h u m a n monocytes. Scand. J. Immunol. 17, 403, 1983.
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JOHNSON, E., B~GWALD, J. and SELJELID, R. E v i d e n c e that a g a r o s e m u s t be i n t e r n a l i z e d to s t i m u l a t e m o u s e m a c r o p h a g e s in vitro. Scand. J. Immunolm 16, 525, 1982.
10. JOHNSON, E., ESKELAND, T. and B E R T H E U S S E N , K. Phagocytosis by h u m a n m o n o c y t e s of p a r t i c l e s a c t i v a t i n g the a l t e r n a t i v e p a t h w a y of complement. Scand. J. Immunol. 19, 31, 1984. ll. JOHNSON, mediated phages. C3bi and 18, 193,
E. and ESKELAND, T. C o m p l e m e n t (C3)-receptorp h a g o c y t o s i s of a g a r o s e b e a d s by m o u s e m a c r o I. I n t r a c e l l u l a r d e g r a d a t i o n og a g a r o s e - b o u n d C3b by l y s o s o m a l enzymes. Scand. J. Immunol. 1983.
12. JOHNSON, E. and ESKELAND, T. C o m p l e m e n t (C3)receptorm e d i a t e d p h a g o c y t o s i s of agarose b e a d s by m o u s e m a c r o phages. II. E x t r a c e l l u l a r a c t i v a t i o n of m a c r o p h a g e d e r i v e d c o m p l e m e n t on agarose via the a l t e r n a t i v e pathway. Scand. J. Immunol. 18, 169, 1983. 13. JOHNSON, E., SLEJELID, R., B#GWALD, J., LARM, O. and SCHOLANDER, E. E n d o c y t o s i s of a g a r o s e in 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 in vitro. Scand. J. Immunol. 15, 205, 1982. 14. LACHMANN, P.J., OLDROYD, R.G., M I L S T E I N , C. and WRIGHT, B.W. Three rat m o n o c l o n a l a n t i b o d i e s to C3. I m m u n o l o g y 41, 503, 1980. 15. MANN, J., O'BRIEN, R., H O S T E T T E R , M.K., ALPER, C.A. ROSEN, F.S. and BABIOR, B.M. The third c o m p o n e n t of complement: c o v a l e n t a t t a c h e m e n t of a r a d i o a c t i v e sugar to the labile b i n d i n g site of C3 via the a l t e r n a t i v e pathway. J. Immunol. 370, 1981. 16. M U N T H E - K A A S , A.C. and KAPLAN, G. E n d o c y t o s i s by m a c r o p h a ges. pp. 19-55 in Carr, I. and Daems, W.T. (eds.). The R e t i c u l o e n d o t h e l i a l System. Vol. I, 1980. 17. ROSS, G.D. A n a l y s i s of the d i f f e r e n t types of l e u k o c y t e m e m b r a n e c o m p l e m e n t r e c e p t o r s and their i n t e r a c t i o n with the c o m p l e m e n t system. J. Immunol. M e t h o d s 37, 197, 1980. 18. SELJELID, R., B~GWALD, J. and L U N D W A L L , A. lation of m a c r o p h a g e s in vitro. Exp. Cell. 1981.
G l y c a n stimuRes. 131, 121,
19. WRIGHT, S.D. and SILVERSTEIN, S.C. Tumor-promoting phorbol esters s t i m u l a t e C3b and C3bi receptor-media, ted p h a g o cytosis in c u l t u r e d h u m a n m o n o c y t e s . J. Exp. Med. 156, 1149, 1982. Received : January, 1984 Accepted : March, 1984
A T T A C H M E N T AND P H A G O C Y T O S I S
BY SALMON M A C R O P H A G E S
OF A G A R O S E BEADS C O A T E D WITH HUMAN C3b and C3bi Egil J o h n s o n and P a t r i c k Smith Institute of M e d i c a l Biology, D e p a r t m e n t of M o r p h o l o g y I, U n i v e r s i t y of Troms@, N - 9 0 0 1 Troms~, N o r w a y
ABSTRACT
A g a r o s e beads (diameter 5-10 ~m) p r e i n c u b a t e d in h u m a n serum b e c a m e a s s o c i a t e d (attached and ingested) to 50-60% of the salmon m a c r o p h a g e s w i t h i n 60 m i n u t e s However, Beads p r e i n c u b a t e d in serum t r e a t e d w i t h h e a t i n g (50°C, 20 min) or w i t h EDTA (i0 mM) to inhibit the a c t i v a t i o n of a l t e r n a t i v e c o m p l e m e n t pathway, were not a s s o c i a t e d to the p h a g o c y t e s . F u r t h e r m o r e , a g a r o s e b e a d s c o a t e d w i t h h u m a n C3b and C3bi after i n c u b a t i o n w i t h i s o l a t e d c o m p l e m e n t factors (C3, D, B), were a s s o c i a t e d to 30-40% of the phagocytes. A b o u t 80% of the c e l l - a s s o c i a t e d a g a r o s e beads was i n t r a c e l l u l a r l y located. C o n v e r s i o n by t r y p s i n t r e a t m e n t (0.01%) of a g a r o s e b o u n d C3bi to C3d, a b o l i s h e d the a s s o c i a t i o n of such beads to the m a c r o p h a g e s . The results d e m o n s t r a t e that salmon m a c r o p h a g e s p o s e s s c o m p l e m e n t r e c e p t o r s that bind h u m a n C3b and C3bi. A g a r o s e beads c o a t e d with these ligands (C3b and C3bi) are a t t a c h e d and i n g e s t e d by the phagocytes.
INTRODUCTION We have p r e v i o u s l y shown that the g l y c a n (polysaccharide) agarose s t i m u l a t e m o u s e m a c r o p h a g e s in v i t r o as m e a s u r e d by i n c r e a s e d 1 ~ C - g l u c o s a m i n e i n c o r p o r a t i o n into m a c r o m o l e c u l e s 623
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r e d u c e d 5 " - n u c l e o t i d a s e activity, i n c r e a s e d cell spreading (13,18) and c y t o t o x i c e f f e c t on tumor cells (9). We looked into the m e c h a n i s m b e h i n d the s t i m u l a t o r y e f f e c t of agarose on the m a c r o p h a g e s . Our main finding was that agarose must be e n d o c y t o s e d by the m a c r o p h a g e s in order to exert its stimulatory e f f e c t (9). A g a r o s e like other s t i m u l a t o r y glycans, are p o t e n t a c t i v a t o r s of the a l t e r n a t i v e p a t h w a y of c o m p l e m e n t (13,18). We found that agarose must be c o a t e d with C3b or C3bi before a t t a c h m e n t and u p t a k e of the g l y c a n by the c o r r e s p o n d i n g r e c e p t o r s for C3b and C3bi on both m o u s e m a c r o p h a g e s (8,11,12) and human m o n o c y t e s (8,10). F r o m this it was clear that c o m p l e m e n t c o a t e d agarose beads d i a m e t e r (5-10 ~m) was a novel and e x c e l l e n t p a r t i c l e for s t u d y i n g the o c c u r e n c e of c o m p l e m e n t r e c e p t o r s on m o n o n u clear phagocytes. The c o m p l e m e n t s y s t e m and c o m p l e m e n t receptors on m o n o n u c l e a r p h a g o c y t e s are e s s e n t i a l for the p r o t e c t i o n of the host a g a i n s t m i c r o o r g a n i s m s . T h e r e f o r e , it is of biological i n t e r e s t to i n v e s t i g a t e w h e t h e r p h a g o c y t e s further down in the animal k i n g d o m p o s e s s receptor(s) for c o m p l e m e n t factor C3, and w h e t h e r human c o m p l e m e n t p o s s i b l y can interact with such receptors. We found that salmon m a c r o p h a g e s p o s e s s r e c e p t o r s for h u m a n C3b and C3bi. A g a r o s e beads c o a t e d with these ligands are a t t a c h e d and i n g e s t e d by the p h a g o c y t e s .
_MATERIALS AND M E T H O D S E x p e r i m e n t a l fish. Salmon (10-20 g) from fresh w a t e r was o b t a i n e d from J . E . P e t t e r s e n , B r e e d i n g Station, K v a l ~ y v~gen, Norway, and kept in tanks of d e c h l o r i n a t e d water at 14-16oc until use. While in the l a b o r a t o r y the fish were fed w i t h Tess salmon food. C u l t u r e medium. The m e d i u m used for p r e p a r a t i o n of cell s u s p e n s i o n and m a i n t e n a n c e in c u l t u r e was L-15 (Leibovitz, G r a n d Island, B i o l o g i c a l Company, U.S.A.) s u p p l e m e n t e d with 0.033 g g l u c o s e / m l , 100 IU p e n i c i l l i n - s t r e p t o m y c i n / m l (Gibco Biocult, Scotland) and 10% heat i n a c t i v a t e d (56°C, 30 min) foetal calf serum (FCS) (Gibco). M e d i u m was a d j u s t e d to pH 7.6 and m O s m of 320. P r e p a r a t i o n of l y m p h o i d cells. The salmon m a c r o p h a g e s were i s o l a t e d largely as p r e v i o u s l y d e s c r i b e d (5). However, there are m i n o r m o d i f i c a t i o n s r e g a r d i n g the d e n s i t i e s of the p e r c o l l g r a d i e n t s used, and the m e t h o d will be d e s c r i b e d in detail. Fish were k i l l e d by d e c a p i t a t i o n . The p r o n e p h r o s was d i s s e c t e d out by v e n t r a l incision, c a r e f u l l y lifted out w i t h c u r v e d forceps and t r a n s f e r r e d to 3 ml L-15 medium. The cell s u s p e n s i o n was p r e p a r e d by t e a s i n g the p r o n e p h r o s with two bent glass rods over a s t a i n l e s s steel m e s h (diameter 0.03 mm) in a small glass dish. Clumps of cells were r e m o v e d after s e d i m e n t a t i o n in a test tube (Falcon, C o c k e y s v i l l e , U.S.A.),
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and the cells r e m a i n i n g in s u s p e n s i o n were c o u n t e d and their v i a b i l i t y t e s t e d by t r y p a n blue e x c l u s i o n (0.05% solution). D i f f e r e n t i a l c e n t r i f u ~ a t i o n of l y m p h o i d cells. The cell s u s p e n s i o n was loaded onto Percoll g r a d i e n t s (Pharmacia Fine Chemicals, Uppsala, Sweden) in 25 ml tubes (Nunco) for d e n s i t y centrifugation. Percoll was d i l u t e d in L-15 to give the diffe r e n t g r a d i e n t d e n s i t i e s of 1.060, 1.070 and 1.090, respectively. The g r a d i e n t s w e r e c a r e f u l l y l a y e r e d from the b o t t o m of the tubes as follows: 1.090 (2.5 ml), 1.070 (5.0 ml) and 1.060 (2.5 ml). Then the cell s u s p e n s i o n in L-15 (3.0 ml) was g e n t l y l a y e r e d on top of the g r a d i e n t s and the tube was c e n t r i fuged at 400 g in a D a m o n / I E C D i v i s i o n c e n t r i f u g e Pr-6000. The cell b a n d c o n t a i n i n g 80-90% m a c r o p h a g e like cells plus some l y m p h o c y t e s and p o l y m o r p h o n u c l e a r g r a n u l o c y t e s , was i d e n t i f i e d b e t w e e n the upper two g r a d i e n t s in the tube. In v i t r o c u l t u r e conditions. The cell s u s p e n s i o n from the u p p e r cell b a n d was w a s h e d in L-15 m e d i u m s u p p l e m e n t e d w i t h FCS and counted. A b o u t 0.8 x 106 c e l l s / m l were seeded on glass c o v e r s l i p s (diameter 14 mm) in C O s t a r tissue c u l t u r e p l a t e s (Costar, C a m b r i d g e , Mass., U.S.A.). The cells were c u l t u r e d at 10°C in air and w a s h e d three times in L-15 after 2 hours of i n c u b a t i o n to r e m o v e n o n - a d h e r e n t cells, and f u r t h e r c u l t u r e d in L-15 w i t h FCS. All e x p e r i m e n t s were p e r f o r m e d after 3 days of i n c u b a t i o n u n d e r s e r u m f r e e conditions, after w a s h i n g to r e m o v e FCS. In 3 days old c u l t u r e s about 90-98% of the cells w e r e well s p r e a d and c a p a b l e of p h a g o c y t o s i s (fiwed sheep e r y t h r o c y t e s , h e a t k i l l e d C a n d i d a and living Yeast cells) (5), w h i c h are c r i t e r i a for the i d e n t i f i c a t i o n of m a c r o p h a g e s . H u m a n serum. Human b l o o d was o b t a i n e d by v e n i p u n c t u r e of h e a l t h y young donors and a b s o r b e d by p a s s a g e t h r o u g h a S e p h a r o s e 4B (Pharmacia) c o l u m n at 4°C to r e m o v e C - r e a c t i v e p r o t e i n (ii) and any a n t i b o d i e s a g a i n s t a g a r o s e (8). P r e i n c u b a t i o n of a ~ a r o s e beads in h u m a n serum. Agarose beads (diameter 5-10 Hm, 1 mg = 6 x 106 beads) was a gift from P h a r m a c i a Fine Chemicals, Uppsala, Sweden. The a g a r o s e beads at a c o n c e n t r a t i o n of 1 mg/ml were i n c u b a t e d in h u m a n serum d i l u t e d 1:2 for 15 m i n u t e s at 37oc, and then w a s h e d three times in L-15 by c e n t r i f u g a t i o n at 450 g for 5 minutes. The beads w e r e n o w m a i n l y c o a t e d w i t h C3bi (8,11,15). P r e p a r a t i o n of a @ a r o s e beads c o a t e d w i t h C3b and C3bi. The m e t h o d of c o a t i n g a g a r o s e beads w i t h h u m a n C3b and C3bi by i n c u b a t i o n w i t h t r y p s i n and f u n c t i o n a l l y pure c o m p l e m e n t f a c t o r s of the a l t e r n a t i v e p a h t w a y (C3, B, D) has been d e s c r i bed (8,11). The i n c u b a t i o n of the beads w i t h B, D and C3 was r e p e a t e d five times. By also using ~ 2 S I - l a b e l l e d (6) C3 to a s p e c i f i c a c t i v i t y of 0.3 H C i / ~ , the a m o u n t of 12sI on 1 ~g a g a r o s e beads was a b o u t 5 x i0 J c.p.m, g i v i n g a b o u t 1.7 Hg p r o t e i n per 100 ~g a g a r o s e (ii). One half of the C3b c o a t e d a g a r o s e beads was t r e a t e d with h u m a n serum d i l u t e d 1:20 in g e l a t i n e - b a r b i t a l b u f f e r (GVB) c o n t a i n i n g i0 mM e t h y l e n e d i a minetetraacetate (EDTA) for 15 m i n u t e s to c o n v e r t a g a r o s e
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l i n k e d C3b to C3bi. A b o u t 83% of a g a r o s e b o u n d C,3b w a s c o n v e r ted to C3bi, as p r e v i o u s l y s h o w n w i t h the use of rat m o n o c l o n a l a n t i - h u m a n C3c and - C 3 d a n t i b o d i e s (8,14). A s s o c i a t i o n of a ~ a r o s e b e a d s to the m a c r o p h a ~ e s . Beads w e r e a d d e d to t h e p h a g o c y t e s at a b e a d to cell r a t i o of 3:1. A f t e r i n c u b a t i o n for 60 m i n u t e s at 10°C the c e l l s w e r e w a s h e d c a r e f u l l y t h r e e t i m e s by d i p p i n g the c o v e r s l i p s in w a r m L-15 to r e m o v e free a g a r o s e b e a d s , a n d t h e n f i x e d in 2.5% g l u t a r a l d e h y d e , 0.i M s u c r o s e in 0.i M c a c o d y l a t e b u f f e r , pH 7.3. Association ( a t t a c h m e n t and i n g e s t i o n ) of a g a r o s e b e a d s to the p h a g o c y t e s was e x a m i n e d in a p h a s e c o n t r a s t m i c r o s c o p e (Carl Zeiss) and d e f i n e d as the p e r c e n t a g e of the t o t a l cell p o p u l a t i o n a s s o c i a t e d w i t h at l e a s t one a g a r o s e bead. The dist i n c t i o n b e t w e e n a t t a c h m e n t and i n g e s t i o n was e v a l u a t e d by o b s e r v i n g m i c r o s c o p i c a l l y w h e t h e r the o u t e r c e l l m e m b r a n e e n c l o s e d the e n t i r e a g a r o s e bead. Micro~raphs. The macrophage cultures were fixed m i c r o g r a p h s w e r e t a k e n in a p h a s e c o n t r a s t m i c r o s c o p e .
before
RESULTS Cell-association of a @ a r o s e b e a d s p r e i n c u b a t e d in h u m a n s e r u m or c o a t e d w i t h C 3 b or C3bi. S i n c e a g a r o s e is an a c t i v a t o r of the a l t e r n a t i v e p a t h w a y of c o m p e l e m e n t , i n c u b a t i o n in s e r u m i n i t i a l l y g i v e s a g a r o s e l i n k e d C3b. H o w e v e r , a f t e r 15 m i n u t e s of i n c u b a t i o n the b e a d s a r e m a i n l y c o a t e d w i t h C 3 b i (ii,12,15). S u c h b e a d s w e r e a d d e d to the m a c r o p h a g e s for 60 m i n u t e s , and 5 0 - 6 0 % of the t o t a l c e l l p o p u l a t i o n w a s a s s o c i a ted to the a g a r o s e b e a d s (data n o t s h o w n ) . U s u a l l y the p h a g o c y t e s w e r e a s s o c i a t e d to o n e b e a d , o c c a s i o n a l l y two b e a d s . A g a r o s e b e a d s p r e i n c u b a t e d in h u m a n s e r u m t r e a t e d w i t h i0 ~ 4 E D T A or w i t h h e a t i n g (50oc, 20 min) as w e l l as n a t i v e a g a r o s e b e a d s , w e r e n o t a s s o c i a t e d to the m a c r o p h a g e s (Table I) . TABLE Association
of
serum
I
treated
T r e a t m e n t of aHarose beads
a@arose
beads
to m a c r o p h a g e s
x Association
Human serum (15 min, 37°C) Human serum (15 min, 37oc) E D T A (I0 mM) 50°C, 20 m i n None x
+ = Association (attachment - = no a s s o c i a t i o n
and
ingestion),
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A b o u t 30-40% of the total cell p o p u l a t i o n w a s a s s o c i a t e d to the b e a d s c o a t e d w i t h C3b or C3bi (Figures 1 a) and 1 b)). As e v a l u a t e d by p h a s e c o n t r a s t m i c r o s c o p y , a b o u t 80% of the a g a r o s e ( p r e i n c u b a t e d in serum, c o a t e d w i t h C3b or C3bi) was i n t r a c e l l u l a r l y l o c a t e d (Figures 1 a) and 1 b)), w h i c h is in a c c o r d a n c e w i t h c o m p a r a b l e s t u d i e s on h u m a n m o n o c y t e s (I0) and m o u s e m a c r o p h a g e s (ii). The p h a g o c y t e s b e c a m e less s p r e a d and p a r t l y d e t a c h e d f r o m the g l a s s c o v e r s l i p s , a f t e r i n g e s t i o n of the a g a r o s e b e a d s (Figures 1 a) a n d 1 b)). T r y p s i n t r e a t m e n t (0.01% for 30 m i n u t e s a t 37°C) of C3bi c o a t e d a g a r o s e beads to c o n v e r t C3bi to m a i n l y C 3 d (8), abolished the a s s o c i a t i o n of such b e a d s to the p h a g o c y t e s (Table II). However, t r y p s i n i z a t i o n of a g a r o s e l i n k e d C3b did not r e d u c e a s s o c i a t i o n of these b e a d s to the m a c r o p h a g e s , since C3b is r e l a t i v e l y r e s i s t a n t to short e x p o s u r e s of t r y p s i n u n d e r these c o n d i t i o n s (8). TABLE Association
of c o m p l e m e n t
Agarose beads coated with C3b C3b C3bi C3bi
coated
II a~arose
Treatment None Trypsin None Trypsin
(0.01%) (0.01%)
a)
beads
to m a c r o p h a g e s
Association + + + -
b) FIG
I
Phase c o n t r a s t m i c r o g r a p h s (x 400) of s a l m o n m a c r o p h a g e s . A g a r o s e beads c o a t e d w i t h C 3 b (a) and C3bi (b) are a s s o c i a t e d to m a n y of the p h a g o c y t e s . The beads are i n d i c a t e d by b l a c k arrows.
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DISCUSSION We p r e s e n t e v i d e n c e that salmon m a c r o p h a g e s p o s e s s comp l e m e n t r e c e p t o r s that bind h u m a n c o m p l e m e n t factors C3b and C3bi. F u r t h e r m o r e , that c o m p l e m e n t (C3b or C3bi) c o a t e d a g a r o s e beads are p h a g o c y t o s e d by the phagocytes. A s s o c i a t i o n of serum t r e a t e d beads was d e p e n d e n t on an i n t a c t f u n c t i o n of the a l t e r n a t i v e c o m p l e m e n t p a h t w a y (Table I), since i n a c t i v a t i o n of factor B (50°C, 20 minutes) or p r e s e n c e of E D T A (!0 ~M) a b o l i s h e d the a s s o c i a t i o n of the beads to the cells. This m e a n s that a c t i v a t i o n of the a l t e r n a t i v e p a t h w a y of c o m p l e m e n t is a p r e r e q u i s i t e for the a s s o c i a t i o n of the a g a r o s e to the m a c r o p h a g e s . The i n c r e a s e d a s s o c i a t i o n (20-30%) to the p h a g o c y t e s of serum t r e a t e d beads c o m p a r e d to C3b and C3bi c o a t e d beads, is p r o b a b l y m a i n l y due to more c o m p l e m e n t (mainly C3bi) (8,11,15) on the former beads. However, this d i f f e r e n c e may also be r e l a t e d to b i n d i n g of a d d i t i o n a l serum p r o t e i n s to the beads, e s p e c i a l l y C5b (ii) and f i b r o n e c t i n w h i c h i n t e r a c t s with factor C3 (7). C3b and C3bi c o a t e d agarose b e a d s a s s o c i a t e d e q u a l l y to the p h a g o c y t e s , even though C3bi has h i g h e r a f f i n i t y for the C3bi r e c e p t o r than C3b for the C3b r e c e p t o r (8,10,19). Thus our finding is m o s t likely c a u s e d by the large a m o u n t of agarose linked c o m p l e m e n t (1.7 ~g p r o t e i n per i00 ~g agarose), w h i c h d i m i n i s h the s i g n i f i c a n e of p r o s p e c t i v e d i f f e r e n c e s in a f f i n i t i e s b e t w e e n the ligands and their c o r r e s p o n d i n g receptors. A n o t h e r p o s s i b i l i t y is that the salmon m a c r o p h a g e s only p o s e s s a single c o m p l e m e n t C3 r e c e p t o r that can not d i s t i n q g u i s h b e t w e e n human C3b and C3bi due to the species incompatibility. However, this e x p l a n a t i o n is less likely c o n s i d e r i n g the separate r e c e p t o r s for C3b and C3bi r e p o r t e d (1,8,10,17,19) on b o t h m o u s e m a c r o p h a g e s and h u m a n m o n o c y t e s . T r y p s i n treatm e n t of the C3bi c o a t e d a g a r o s e beads (Table II), w h i c h r e m o v e d a b o u t 83% of a g a r o s e b o u n d C3c (8), a b o l i s h e d the a s s o c i a t i o n of such beads (mainly C3d coated) to the p h a g o c y t e s , w h e r e a s the t r y p s i n r e s i s t a n t C3b c o a t e d beads still a s s o c i a t e d to the cells. These r e s u l t s c l e a r l y d e m o n s t r a t e that the a t t a c h m e n t of c o m p l e m e n t c o a t e d a g a r o s e to the C3b and C3bi receptors, is m e d i a t e d via the C3c p o r t i o n of the C3 molecule. The k i n e t i c s of the i n g e s t i o n of agarose by the phagocytes was not s t u d i e d in detail. However, we c o n c l u d e that the u p t a k e r e a c h e d a p l a t e a u w i t h i n one hour, since longer incubation time did not increase the a m o u n t of beads a s s o c i a t e d to the cells (data not shown). The general c o n c e p t is (4,16,19) that r e s i d e n t m o n o n u clear p h a g o c y t e s (mouse m a c r o p h a g e s , human monocytes) only bind C3b and C3bi c o a t e d sheep e r y t h r o c y t e s , w h i c h are not i n g e s t e d u n l e s s the p h a g o c y t e s are s t i m u l a t e d with specifig agents. On the contrary, this study and p r e v i o u s results (8,10,11) have shown that n o n o n u c ! e a r p h a g o c y t e s ingest comp l e m e n t c o a t e d a g a r o s e beads i n d e p e n d e n t of s p e c i f i c stimulation. This d i s c r e p a n c y in r e s u l t s m u s t be r e l a t e d to the d i f f e r e n t n a t u r e of the test p a r t i c l e s being used (agarose
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beads v e r s u s sheep e r y t h r o c y t e s ) . F u r t h e r m o r e , we found (10, ii) a c o r r e l a t i o n b e t w e e n i n g e s t i o n of test p a r t i c l e s by the p h a g o c y t e s and their a b i l i t y to a c t i v a t e the a l t e r n a t i v e pathway of complement. R e c e n t l y it is also r e p o r t e d that e c h i n o i d p h a g o c y t e s (2, 3) bind and i n g e s t sheep e r y t h r o c y t e s c o a t e d with human C3bi. The wide d i s t r i b u t i o n of c o m p l e m e n t like r e c e p t o r s in mammals, fish and i n v e r t e b r a t e s , e m p h a s i z e the s i g n i f i c a n c e of complem e n t as a host s y s t e m a g a i n s t m i c r o o r g a n i s m s .
ACKNOWLEDGEMENTS This work was s u p p o r t e d by g r a n t s from The N o r w e g i a n R e s e a r c h C o u n c i l for S c i e n c e and the H u m a n i t i e s and the N o r w e g i a n F i s h e r i e s R e s e a r c h Council.
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19. WRIGHT, S.D. and SILVERSTEIN, S.C. Tumor-promoting phorbol esters s t i m u l a t e C3b and C3bi receptor-media, ted p h a g o cytosis in c u l t u r e d h u m a n m o n o c y t e s . J. Exp. Med. 156, 1149, 1982. Received : January, 1984 Accepted : March, 1984