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In vitro agglutinin production by earthworm leukocytes
DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. 12, pp. 531-547, ]988 0145-305X/88 $3.00 .+ Printed in the USA Copyright (c) 1988 Pergamon Press plc All rights reserved
IN VITRO A G G L U T I N I N P R O D U C T I O N
BY EARTHWORM L E U K O C Y T E S
Elizabeth A. Stein and Edwin L. Cooper D e p a r t m e n t of Anatomy, School of Medicine, U n i v e r s i t y of California, Los Angeles, California
ABSTRACT
Leukocytes of the earthworm, Lumbricus terrestris, s e c r e t e a g g l u t i n i n s in vitro, as shown by measuring agglutinin titers of the culture medium and by observing secretory rosette formation by l e u k o c y t e s with erythrocytes. Leukocytes form the highest percentages of secretory rosettes with rabbit e r y t h r o c y t e s (RBC) and with other RBC species in the o r d e r : rat, g u i n e a pig, m o u s e , c a l f , s h e e p , h o r s e , goat. L e u k o c y t e s d i s p l a y e d a l l o t y p i c s p e c i f i c i t y by f o r m i n g rosettes s e l e c t i v e l y with e r y t h r o c y t e s from different individual rabbits. Eight sugars inhibited rosette formation, along with the p o l y s a c c h a r i d e mannan and the g l y c o p r o t e i n s t h y r o g l o b u l i n and bovine subm a x i l l a r y mucin. C y c l o h e x a m i d e did not affect rosette formation, s u g g e s t i n g that a g g l u t i n i n s may be p r e f o r m e d and stored in leukocytes prior to secretion. Leukocytes also formed E-type rosettes with erythrocytes, but a p p a r e n t l y utilized d i f f e r e n t receptors from those of s e c r e t o r y rosettes since they were not inhibited by the same sugars. INTRODUCTION
Hemocytes or l e u k o c y t e s of s e v e r a l i n v e r t e b r a t e s c o n t a i n agglutinins and presumably secrete them (1-4). However, agglutinin production has rarely been observed in vitro. R o s e t t e - f o r m i n g cells have been d e s c r i b e d in e a r t h w o r m s (5-8), insects (9) and tunicates (I0), but in most instances these have been E, type rosettes. Secretory rosettes have been d e s c r i b e d in L u m b r i c u s terrestris (7,11) and Styela clava (I0) and are formed 531
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by a g g l u t i n i n - p r o d u c i n g cells. We show here that Lumbricus leukocytes can secrete a g g l u t i n i n s in vitro, by: I) direct assays of a g g l u t i n i n s in the c u l t u r e m e d i u m ; and 2) q u a n t i f y i n g s e c r e t o r y r o s e t t e - f o r m i n g cells. A s e c o n d c e l l type, the c h l o r a g o g e n cell, c o n t a i n s agglutinins, but because of their fragility have not been cultured in vitro. Lumbricus leukocytes exhibit s p e c i f i c i t y in their reactions with erythrocytes; the p e r c e n t a g e s of s e c r e t o r y and E-type rosettes vary, d e p e n d i n g on the e r y t h r o c y t e species. It is of i n t e r e s t t h a t l e u k o c y t e s a l s o r e c o g n i z e a l l o t y p i c differences, forming secretory rosettes specifically with erythrocytes from d i f f e r e n t i n d i v i d u a l s of the same s p e c i e s . Assays using secretory rosettes now provide a tool for analyzing the p r o d u c t i o n of i n v e r t e b r a t e a g g l u t i n i n s and for a s s a y i n g humoral immune responses at the c e l l u l a r level.
M A T E R I A L S AND METHODS
Earthworms: Adult earthworms (Lumbricus terrestris) were purchased from Golden West Cricket Co., Paramount, CA and m a i n t a i n e d at 1 5 ° C in B u s s B e d d i n g ( B u s s M a n u f a c t u r i n g Co., Lanark, IL) with supplemental feedings of dry baby cereal. Agglutinin content in Iysates of leukocytes: Agglutinin levels were measured {tom two cell sources, l e u k o c y t e s and c h l o r a g o g e n cells. Leukocytes, c o n t a i n e d in coelomic fluid, were c o l l e c t e d by inserting a sharpened Pasteur pipette into the coelomic cavity posterior to the c l i t e l l u m and allowing it to fill with coelomic fluid by intra-coelomic pressure. The fluid was c e n t r i f u g e d at I00 g, s u p e r n a t a n t removed and l e u k o c y t e s washed three timesli~0 normal (0.85%) saline. Cells were counted, a d j u s t e d to 1 x cells/ml and l y s e d by a s o n i c d i s r u p t e r . The l y s a t e was c e n t r i f u g e d at 10,000g for 30 min to remove particulate matter and the supernatant retained for assays. A g ~ l u t i n i n content in Iysates of c h l o r a g o g e n cells: C h l o r a g o g e n c e l l s w e r e c o l l e c t e d by a n e s t h e t i z i n g w o r m s for 1 hr in 5% ethanol in water (v/v) then cutting them open longitudinally. Chloragogen cells, which invest the outer surface of the digestive t r a c t , w e r e g e n t l y s c r a p e d off w i t h a s c a l p e l and suspended in 0.85% saline. Since cells were disrupted, it was i m p o s s i b l e to p e r f o r m c e l l c o u n t s ; h o w e v e r , s u s p e n s i o n s w e r e checked microscopically to i n s u r e that t h e r e was n e g l i g i b l e c o n t a m i n a t i o n by leukocytes. Cell samples were collected from 12 worms, as three samples from four worms. The cell suspension was lysed by sonic d i s r u p t i o n and c e n t r i f u g e d as for leukocytes to remove particulate matter. Leukocyte and chloragogen cell lysates were assayed for a g g l u t i n a t i o n titers using rabbit erythrocytes (RRBC) (see below) and for protein c o n c e n t r a t i o n s by a modified Lowry procedure (12). ~eqkocyte collection for in vitro cell cultures: Earthworm coelomic fluid, c o n t a i n i n g leukocytes, was removed a s e p t i c a l l y as p r e v i o u s l y d e s c r i b e d (13). Briefly, worms were a n e s t h e t i z e d as d e s c r i b e d a b o v e , then g e n t l y , but r a p i d l y s c r u b b e d w i t h 95% ethanol to reduce external contamination, and rinsed in sterile 0.85% saline. Worms were then placed in sterile petri dishes and cut o p e n l o n g i t u d i n a l l y on the d o r s a l s u r f a c e to e x p o s e the
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coelomic cavity. C o e l o m i c fluid and l e u k o c y t e s were washed out with sterile saline, c o l l e c t e d by sterile Pasteur pipettes and c e n t r i f u g e d for 5 min at 150 g. The supernatant was removed and cells w a s h e d twice in saline. Assays of in vitro ag@lutinin production: Leukocytes were r e s u s p e n d e d in a modified L i e b o v i t z - 1 5 (L-15) medium consisting of: L-15 medium c o n t a i n i n g L-glutamine, 60% (v/v); Lumbricus b a l a n c e d salt solution (LBSS) (14), 40%: gentamycin, 5 mg/100 ml. Fetal calf serum used p r e v i o u s l y in e a r t h w o r m culture media (33), was not added, since it inhibited coelomic fluid a g g l u t i n a t i o n of erythrocytes, and did not increase leukocyte v i a b i l i t y in our assays. The pH was a d j u s t e d to 7.2 and the medium sterilized by filtration. L e u k o c y t e d e n s i t y was adjusted to 1 x i0 cells/ml and the s u s p e n s i o n added as 0.2 ml a l i q u o t s to wells of sterile, 96-well, flat-bottom culture plates (Costar Plastics, M~A Bioproducts, Los Angeles, CA). Cultures were m a i n t a i n e d at 15 C and monitored periodically for contamination. At specific intervals, contents of the wells were removed, centrifuged to remove cells and p a r t i c u l a t e matter, and the supernatant assayed for a g g l u t i n i n titers. In vitro a ~ l u t i n i n p r o d u c t i o n in the presence of erythrocytes: A separate set of experiments was designed to determine if agglutinin i n d u c t i o n w o u l d o c c u r in v i t r o , as we p r e v i o u s l y demonstrated in vivo (15). ~eukocyte concentrations were adjusted before plating to 2 x I0v cells/ml and an equal volume of 0 . 1 2 5 % (v/v) R R B C s u s p e n s i o n (see b e l o w ) was a d d e d . The suspension of mixed leukocytes and RRBC was plated as before in sterile m i c r o t i t e r plates at 0.2 ml/well. Fluid was removed from the wells at specific intervals and titers of the supernatant determined. T i t e r s of s e c r e t e d a g g l u t i n i n : F i f t y m i c r o l i t e r s of c u l t u r e supernatant, either from wells c o n t a i n i n g leukocytes alone or l e u k o c y t e s plus RRBC, were added to wells of U-type m i c r o t i t e r plates. The fluid was serially d i l u t e d with p h o s p h a t e - b u f f e r e d saline (PBS) and 25 1 of a 2% (v/v) RRBC suspension in PBS added to each well. Plates were shaken gently for several minutes, then a l l o w e d to stand for 1 hr. Titers were read and recorded as the r e c i p r o c a l of the highest d i l u t i o n showing agglutination. Erythrocytes: R a b b i t b l o o d was c o l l e c t e d in h e p a r i n f r o m the marginal ear vein and washed three times in 0.85% saline. The buffy coat was removed and e r y t h r o c y t e s (RRBC) r e - s u s p e n d e d at a d e n s i t y of 2% (v/v) in LBSS that had been adjusted with NaCI to 300mOsM (LBSS-300). For some experiments, we used a panel of d i f f e r e n t erythrocytes. Guinea pig, calf and goat blood were purchased from Colorado Serum Co., Denver, CO. Sheep and horse b l o o d were purchased from Mission L a b o r a t o r y Supply, Rosemead, CA. Rat and mouse blood were kindly supplied by Dr. Yoko Mullen, Dental Research Institute, University of California at Los Angeles. All types of blood were p r o c e s s e d as for RRBC. Isolation and p r e p a r a t i o n of leukocytes for rosetting: Coelomic f l u i d w a s c o l l e c t e d b y i n s e r t i n g a P a s t e u r p i p e t t e into the c o e l o m i c cavity, as d e s c r i b e d above. The fluid was then mixed gently with cold 0.85% saline and c e n t r i f u g e d at 150 g for i0 min. The s u p e r n a t a n t was removed, cells washed once more and r e s u s p e n d e d in 0.2 ml saline, then mixed with 0.8 ml LBSS-300. By w a s h i n g l e u k o c y t e s i n i t i a l l y in s a l i n e , then r e s u s p e n d i n g l a t e r in L B S S - 3 0 0 , c l u m p i n g was r e d u c e d and l e s s c e l l d a m a g e
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IN VITRO AGGLUTININ PRODUCTION
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o c c u r r e d during resuspension. Cell d e n s i t y was adjusted to I0 6 c e l l s / m l in LBSS-300. O n e - t e n t h ml a l i q u o t s were placed in small tubes and kept at 4°C. L e u k o c y t e s from each worm were c o l l e c t e d and p r o c e s s e d s e p a r a t e l y for all assays. Identification of rosettes: Two percent s u s p e n s i o n s of RBC in L B S S - 3 0 0 w e r e a d d e d as 0.i m l ~ l i q u o t s to an e q u a l v o l u m e of leukocytes in LBSS-300 at i 0 v cells/ml. Leukocytes and e r y t h r o c y t e s were mixed briefly, c e n t r i f u g e d at i00 g for 5 min, then kept at 4vC (see below) for specific time periods. Just before rosettes were to be counted, 0.I ml of a solution of 0.1% crystal violet in LBSS-300 was added to the cell s u s p e n s i o n to stain cell nuclei. Tubes were shaken g e n t l y to r e - s u s p e n d the p e l l e t a n d a s m a l l a m o u n t of the c e l l s u s p e n s i o n p l a c e d in a h e m o c y t o m e t e r chamber. One hundred cells were counted for each sample at each time-point. Fourteen to sixteen worms were used for e a c h set of a s s a y s , each worm providing cells for all time-points. Cells were scored either as s e c r e t o r y rosettes, E-rosettes or non-rosetting cells. Secretory rosettes are leukocytes surrounded by two or more layers of adhering erythrocytes. E-rosettes are leukocytes w i t h f o u r or m o r e erythrocytes adhering to the c e l l s u r f a c e as a s i n g l e l a y e r . N o n - r o s e t t i n g cells c o n s i s t e d of those l e u k o c y t e s with fewer than four a d h e r i n g e r y t h r o c y t e s . Rosette formation was measured with each of the d i f f e r e n t types of erythrocytes: rabbit, rat, mouse, guinea pig, calf, goat and horse. Carbohydrate and g l y c o p r o t e i n inhibition of rosette formation: Sugars and glycoproteins~ as s o l u t i o n s in PBS, w e r e a d d e d to suspensions of Lumbricus leukocytes just before mixing the leukocytes with rabbit RBC. Cultures were scored at 4 hr incubation for s e c r e t o r y and E-type r o s e t t e s as d e s c r i b e d above. The D isomers of glucose, galactose, mannose, glucosamine, galactosamine, mannosamine, N-acetylglucosamine, N-acetylgalactosamine, N-acetylmannosamine, glucuronic acid, -methylmannoside, a n d a r a b i n o s e w e r e used. We a l s o t e s t e d L-fucose, N - a c e t y l n e u r a m i n i c acid and 2 - k e t o - 3 - d e o x y o c t o n a t e (all sugars at 50 mM), the p o l y s a c c h a r i d e mannan (2 mg/ml) and the glycoproteins bovine submaxillary mucin (0.2 mg/ml), and thyroglobulin (2 m g / m l ) . The pH of all s u g a r s o l u t i o n s w a s a d j u s t e d to 7.2. C o n c e n t r a t i o n s were used that had been shown p r e v i o u s l y to inhibit RRBC a g g l u t i n a t i o n (15). Temperature effects on rosette formation: The effects of temperature on r o s e t t e f o r m a t i o n w e r e d e t e r m i n e d in a set of parallel assays p e r f o r m e d at 5°C and 15°C. L e u k o c y t e suspensions were p r e p a r e d and mixed with 2% RRBC s u s p e n s i o n s as d e s c r i b e d above. D u p l i c a t e c u l t u r e s were p r e p a r e d for each time point, one set k e p t at 5 C a n d the o t h e r at 15°C. At s p e c i f i e d times, c u l t u r e s were e x a m i n e d and the s e c r e t o r y and E - r o s e t t e s counted. Rosette formation by l e u k o c y t e s from injected worms: Worms were injected with 0.10-0.15 ml of a 5% s u s p e n s i o n of RRBC in 0.85% saline (15). RRBC were used from two d i f f e r e n t sources, rabbit A (RRBC A) and rabbit M (RRBC M). T w e n t y - f o u r hr later, coelomic fluid was c o l l e c t e d and leukocytes ~rocessed, as d e s c r i b e d above, for assays of rosette formation. Aliquots from each worm were divided into two s e t s ; one set to be a s s a y e d for r o s e t t e formation with erythrocytes from the same rabbit that had supplied the injected RBC, and a second set to be assayed with RBC from a rabbit d i f f e r e n t from the one used for injection. The
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converse experiment was also performed, injecting worms with RRBC from the second source and assaying with both types of RRBC. Cyclohexamide: To determine if suppression of protein synthesis would affect rosette formation, leukocytes and RRBC were cultured in LBSS-300 containing I0 ~ g/ml cyclohexamide (16, 17). Parallel cultures were tested by counting 200 cells, stained with 0.1% trypan blue dye, to determine cell viability in the presence and absence of cyclohexamide. Statistics: Significance levels were determined by the Student's T. test (18). RESULTS Agglutinin levels in leukocytes and chlorago~en cells: Leukocyt~ lysates were assayed and calculated for agglutinin levels per 10cells and per mg protein. Chloragogen cells, since cell counts were not possible, were calculated only as agglutinin units per mg p r o t e i n . L e u k o c y t e s and c h l o r a g o g e n c e l l s b o t h c o n t a i n e d hemagglutinins (Table I). Leukocytes contained 7specific titers of 6.7 units per mg protein and 12.7 units per i0 cells, whereas chloragogen cells contained 41.7 units per mg protein. In vitro a~glutinin production by Lumbricus leukocytes: Leukocytes cultured in nutrient medium r e l e a s e d low levels of hemagglutinins, as shown by measurement of agglutination titers of the supernatant medium. Highest agglutinin levels occurred at 24 and 48 hr, w i t h m e a n t i t e r s of i0.i and 10.2 r e s p e c t i v e l y (Table 2). In those cultures to which rabbit erythrocytes had b e e n a d d e d , a g g l u t i n i n t i t e r s w e r e s l i g h t l y l o w e r than those cultures with leukocytes alone (Table 2). Rosette formation with erythrocytes: Earthworm leukocytes formed b o t h s e c r e t o r y and E - t y p e r o s e t t e s w i t h e r y t h r o c y t e s of all species tested (Table 3). However, the highest percentages of secretory rosettes occurred using RRBC; at 6 hr, 26.7% of the leukocytes formed secretory rosettes (Fig. i). Percentages of E-type rosettes with RRBC ranged from a high of 34.9% at 30 min to 27.1% at 2 hr and rose again slightly at 4 hr to 32.8% (Fig. 2). Using other erythrocytes, the p e r c e n t a g e s of r o s e t t e s varied. With sheep erythrocytes (SRBC), l e u k o c y t e s f o r m e d a maximum of 2.7% secretory rosettes at 2 to 8 hr incubation (Fig. i), and 15.6% E - t y p e r o s e t t e s at 2 hr (Fig. 2). Using other erythrocytes, at 4 hr incubation, secretory rosettes ranged from 0.7% (goat RBC) to 8.5% (rat RBC), and E-type rosettes varied from 8.5% to 15% (Table 3). Carbohydrate and glycoprotein inhibition of rosette formation: Of the 15 sugars tested, 8 inhibited secretory rosette formation to a significant ( P < 0 . 0 5 ) d e g r e e ( T a b l e 4). The p o l y s a c c h a r i d e mannan and the glycoproteins thyroglobulin and bovine submaxillary mucin were also inhibitory. Conversely, E rosette formation was not reduced but instead increased with 8 of the substances, and remained unchanged with the other 3 (Table 4). Amino sugars (glucosamine, galactosamine and mannosamine) were consistently inhibitory, but the acetylated form was inhibitory only for N-acetylgalactosamine. Effects of temperature on rosette formation: Leukocytes incubated at 15-C wi~h RRBC formed secretory rosettes somewhat faster than those at 5-C, with maximum numbers of rosettes occurring at 2-4
536
IN VITRO AGGLUTININ PRODUCTION
TABLE
Agglutinin
Cell
1
L e v e l s of L u m b r i c u s L e u k o c y t e s and C h l o r a g o g e n Cells
type
Per
i0
7
cells
12.7 + 5.5
Chloragogen cells
.
(26)
.
.
.
1.9 + 0.5 .
TABLE
Hours in culture
of
a
5.0
12
+ 0.8
(6)
7.0 + 0.7
(6)
(13)
41.7
(15)
of worms
by L u m b r i c u s
T i t e r s a of p a i r e d l e u k o c y t e b c u l t u r e s , in p r e s e n c e and a b s e n c e of r a b b i t RBC Leukocytes only
6
RBC. Number
6.7
2
In vitro A g g l u t i n i n Leukocytes
A g g l u t i n i n tit~rs of l e u k o c y t e c u l t u r e media
(13)
.
T i t e r s m e a s u r e d by a g g l u t i n a t i o n of r a b b i t S a m p l e mean + s t a n d a r d e r r o r of the mean. given in ( )?
Production
T i t e r a per mg. p r o t e i n (Specific titer)
Protein concentration (mg)
Titer a
Leukocytes
Vol. 12, No. 3
L e u k o c y t e s plus r a b b i t RBC c
24
I0.i
+ 1.5
(20)
4.3
+ 0.6
(9)
3.8 + i.i
(9)
48
10.2 + 2.3
(20)
9.8
+
1.7
(9)
6.7
+
1.5
(9)
(18)
4.5 + 0.8
(9)
2.9
+ 0.6
(9)
72 a
b c
8.4
+ 1.8
Mean + s t a n d a r d e r r o r of the mean. the r ~ c i p r o c a l of the last d i l u t i o n N u m b e r of w o r m s given in ( ). 1 x 106 l e u k o c y t e s
/ml.
0.125%
v/v.
rabbit
RBC,
T i t e r s e x p r e s s e d as showing agglutination.
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IN VITRO AGGLUTININ PRODUCTION
TABLE
537
3
P e r c e n t of L u m b r i c u s L e u k o c y t e s that form S e c r e t o r y and E - r o s e t t e s w i t h D i f f e r e n t T y p e s of E r y t h r o c y t e s (RBC)
Percent
rosettes a
Type of RBC
Secretory M e a n + SEM
E-type M e a n + SEM
rabbit
23.5
+ 1.7
32.8
+ 3.5
(16)
8.5 + 1.0
14.1
+ 1.6
(8)
5.2
15.0
+ 2.7
(5)
8.5 + 1.2
(8)
rat guinea
pig
+ 1.3
N u m b e r of worms
mouse
2.4 + 0.9
sheep
2.7 + I.I
11.2
+ 1.5
(8)
calf
3.2 + 1.3
9.4
+ 1.2
(5)
horse
1.4
+ 0.5
9.6
+ 1.2
(I0)
goat
0.7 + 0.6
14.6
+ 2.3
(i0)
a
After
4 hr.
incubation
at 4°C.
538
IN VITRO AGGLUTININ PRODUCTION
TABLE
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4
I n h i b i t i o n of R o s e t t e F o r m a t i o n by C a r b o h y d r a t e s and G l y c o p r o t e i n s
Percentage Substance a
in N u m b e r s RRBC
Decrease b of S e c r e t o r y
Rosettes
Percent
Increase b
in N u m b e r s RRBC
of
Rosettes
Galatose
54.8
+ 5.5
+26.4
+ 6.2
Galactosamine
54.2
+
+23.4
+
N-acetylgalactosamine
33.1
+ 5.4
Glucuronic
22.3
+
4.9
+16.0
+
Glucosamine
45.2
+ 6.9
+30.7
+ 7.5
Mannosamine
57.6
+
6.2
+29.0
+
55.6
+ 4.9
+32.8
+ 7.4
2-keto-3-deoxyoctonate
52.7
+ 5.1
0
Mannan
22.3
+ 4.8
0
Thyroglobulin
52.4
+
5.7
+46.9
+
26.9
+ 5.2
+41.6
+ 8.4
acid
N-acetylneuraminic
Bovine
submaxillary
acid
mucin
6.1
E
3.2
0 4.4
5.7
7.9
O n l y those s u b s t a n c e s s i g n i f i c a n t l y (P<0.05) i n h i b i t o r y are listed. M o n o s a c c h a r i d e s w e r e used at 50 mM, m a n n a n and t h y r o g l o b u l i n at 2 mg/ml, b o v i n e s u b m a x i l l a r y mucin at 0.2 mg/ml. b
P e r c e n t change, a f t e r 4 hr of c u l t u r e , from n u m b e r s of r o s e t t e s formed in a b s e n c e of i n h i b i t o r , + s t a n d a r d error of mean.
4
culture
Hrs in A
33.4+10.0 3 4 . 3 T 5.7
(9) (8)
Mean
(9) (8)
(I0) (9)
+ standard
29.7+6.8 28.0T4.9
20.4+7.3 30.5T8.1
RRBC M
rosettes b with:
33.2+8.8 (I0) 29.8TII.8 (9)
RRBC
Secretory
24 hrs previously
4 24
24
5
with
deviation.
25.3+5.6 19.0~4.0
25.5+6.8 18.7T7.8
RRBC
A
(9) (8)
(I0) (9)
28.8+3.8 19.8~3.4
22.4+7.8 21.4+5.8
RRBC M
E rosettes b with:
For,nation by Leukocytes from Worms Previously Injected Saline or with Erythrocytes from Rabbit M
bpercent of total leukocytes. Number of worms in ( ).
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26 26 24
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~ Jo
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Fig.
1.
S e c r e t o r y r o s e t t e s formed with rabbit and sheep RBC. Lumbricus leukocytes forming secretory rosettes with r a b b i t and sheep RBC are given as a percent of the total number of leukocytes. X ~ S.D.
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Fig.
2.
E-type r o s e t t e s formed with r a b b i t and sheep RBC. The numbers of leukocytes forming E - r o s e t t e s with rabbit and sheep RBC are given as a percent of the total number of leukocytes. X + S.D.
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26 24 22 20
o ~e ~6 ~4
•
ASSAYED WITH RRSC M
2
~4 HOURS SECRETORY ROSETTES WITH RRBC A AND M (WORMS PREVIOUSLY INJECTED WITH RRBC A)
Fig.
3.
Secretory r o s e t t e s formed w i t h r a b b i t RBC of d i f f e r e n t a l l o t y p e s (RBC A and RBC M). L e u k o c y t e s were taken from w o r m s p r e v i o u s l y i n j e c t e d w i t h RBC from r a b b i t A. X + S.D.
40
35
,.m 30 i.23
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•
ASSAYED WITH RRBC M
a. IO
14 HOURS E-TYPE ROSETTES WITH RRBC A AND M (WORMS PREVIOUSLY INJECTED WITH RRBC A)
Fig.
4.
E-type rosettes f o r m e d w i t h r a b b i t R B C of d i f f e r e n t a l l o t y p e s (RBC A and RBC M). L e u k o c y t e s were taken from w o r m s p r e v i o u s l y i n j e c t e d w i t h RBC from r a b b i t A. X + S.D.
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hr and 4-6 hr, r e s p e c t i v e l y (not illustrated). However, the maximum p e r c e n t a g e s of s e c r e t o r y rosettes were slightly higher for cultures m a i n t a i n e d at 5°C (33%) than those at 15UC (31%). In addition, l e u k o c y t e v i a b i l i t y at 24 hr was slightly higher for cultures m a i n t a i n e d at 5°C (79.5 + 6.4, mean + S.D.) than those at 15°C (72.2 + 8.4). --Rosette formation by leukocytes from injected earthworms. D i s t i n c t i o n b e t w e e n e r y t h r o c y t e a l l o t y p e s : L e u k o c y t e s "{rom worms p r e v i o u s l y injected with RRBC from rabbit A produced d e c r e a s e d numbers of s e c r e t o r y rosettes when tested with e r y t h r o c y t e s from rabbit A (Fig. 3). The p e r c e n t a g e s of RRBC-A s e c r e t o r y rosettes, formed with leukocytes from RRBC-A-injected worms, were approximately 50% lower than normal (Fig. i), and were s i g n i f i c a n t l y lower (p < 0.01) for all time-points from 30 min t h r o u g h 6 hr. At 8 hr--of c u l t u r e , the n u m b e r s of s e c r e t o r y rosettes increased slightly, and at 24 hr the percentage was back to n e a r l y normal levels. Parallel cultures of leukocytes from worms injected with RRBC-A, tested with e r y t h r o c y t e s from rabbit M, produced normal levels of s e c r e t o r y rosettes (approximately 24% at 4 hrs) (Fig. 3). P e r c e n t a g e s of E-type rosettes formed with RRBC-A by leukocytes from worms injected with R R B C - A were somewhat lower than normal but s i g n i f i c a n t l y so only at 30 min; those tested with RRBC-M were at normal levels (Fig. 4). The converse experiment, injecting worms with RRBC-M and m e a s u r i n g rosette formation with RRBC-A and RRBC-M, resulted in a similar r e d u c t i o n of secretory rosettes when tested with RRBC M (Table 5). E rosettes were not s i g n i f i c a n t l y affected. L e u k o c y t e s from worms p r e v i o u s l y injected with saline formed secretory and E rosettes at normal levels when a s s a y e d with both RRBC A and RRBC M (Table 5). Effect of c y c l o h e x a m i d e on rosette fo[mation: The a d d i t i o n of c y c l o h e x a m i d e to the culture m e d i u m did not affect the levels of secretory rosettes w i t h i n the 24-hr period (Fig. 5). Levels of E - r o s e t t e s were slightly, but not significantly, lower in the c y c l o h e x a m i d e - c o n t a i n i n g c u l t u r e s than in regular medium (Fig. 6). V i a b i l i t y of b o t h control and c y c l o h e x a m i d e - t r e a t e d leukocytes decreased e q u a l l y w i t h time. O v e r the 2 4 - h r c u l t u r e p e r i o d , v i a b i l i t y of b o t h s e t s of l e u k o c y t e s was r e d u c e d by a p p r o x i m a t e l y 10%. V a r i a t i o n in v i a b i l i t y was p r i m a r i l y a factor of the p a r t i c u l a r worm from which the leukocytes were harvested, rather than the presence of cyclohexamide. Initial v i a b i l i t y for both sets of cultures varied from 83-97% (X + S.D. = 88.3 + 6.8), d e c r e a s i n g to 7 5 - 8 8 % a t 24 hr (77.4 + 8.5 for c u l t u r e ~ w i t h c y c l o h e x a m i d e , 79.1 + 8.5 for cultures in LBSS alone).
DISCUSSION The coelomic fluid of L u m b r i c u s contains n a t u r a l l y o c c u r r i n g a g g l u t i n i n s against b a c t e r i a and v e r t e b r a t e erythrocytes, and e l e v a t e d a g g l u t i n i n l e v e l s may be i n d u c e d b y i n j e c t i n g t h e s e antigens (15,19). The a g g l u t i n i n s r e a c t w i t h a n u m b e r of v e r t e b r a t e RBC, y i e l d i n g highest titers with rabbit RBC (15). Bacterial a g g l u t i n i n s react with both G r a m - p o s i t i v e and Gramnegative bacteria, and a v a i l a b l e evidence suggests that some of them are identical to the h e m a g g l u t i n i n s (20).
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IN VITRO AGGLUTININ PRODUCTION
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30
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I
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Fig.
5.
Secretory rosettes formed with rabbit RBC in the p r e s e n c e and a b s e n c e of c y c l o h e x a m i d e . Cyclohexamide, I0 g/ml, was continuously present in o n e set of cultures. X + S.D.
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Fig.
6.
E - t y p e r o s e t t e s formed w i t h r a b b i t RBC in the p r e s e n c e a n d a b s e n c e of c y c l o h e x a m i d e . Cyclohexamide, i0 g/ml, was c o n t i n u o u s l y p r e s e n t in one set of c u l t u r e s . X + S.D.
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The source of a g g l u t i n i n s appears to be twofold - l e u k o c y t e s and c h l o r a g o g e n cells. Free c h l o r a g o g e n cells are fragile and not c o m m o n l y found in coelomic fluid, but their cell contents (granules and vesicles) are frequently visible in coelomic fluid or c o n t a i n e d w i t h i n p h a g o c y t i c v a c u o l e s of l e u k o c y t e s (21). Although both types of cells contain agglutinins, c h l o r a g o g e n cells are higher on a per mg protein basis (Table i); however, b e c a u s e of t h e i r f r a g i l i t y , t h e y c a n n o t be e a s i l y c u l t u r e d . L e u k o c y t e s of s e v e r a l o t h e r i n v e r t e b r a t e s h a v e b e e n s h o w n to c o n t a i n agglutinins, including the lobster, Homarus a m e r i c a n u s (I), the cockroach, Leucophaea maderae (2), the silkmoth, Hyalophora cepropia (25), the snail, Lymnaea stagnalis (3) and the oyster, Crassostrea virginica (4). Lumbricus leukocytes secrete small but measurable amounts of a g g l u t i n i n when cultured in vitro. Levels in the culture medium w e r e low ( m a x i m u m t i t e r s of 10.2), w h e n c o m p a r e d to the h i g h titers (up to 1,024) c o m m o n l y found in coelomic fluid from worms that had been previously immunized. Several factors may c o n t r i b u t e to this 100-fold difference: less than optimum culture conditions for l e u k o c y t e s ; lower cell concentration t h a n in coelomic fluid; lack of a c c e s s o r y factors that might be required to stimulate leukocytes to synthesize a n d / o r secrete agglutinins. An attempt to correct this last problem by adding an inducing agent, rabbit RBC, to the c u l t u r e s was not successful. However, since these RBC were visibly a g g l u t i n a t e d after a few hours, they may h a v e a b s o r b e d and r e m o v e d f r o m the f l u i d s o m e of the agglutinins that would otherwise have been available for m e a s u r e m e n t in the assays. R o s e t t e formation is a more sensitive method for d e m o n s t r a t ing a g g l u t i n i n secretion than a g g l u t i n a t i o n titers of culture media. U s i n g r o s e t t i n g , we f o u n d t h a t L u m b r i c u s l e u k c o y t e s secreted a g g l u t i n i n s that reacted with all of the erythrocyte types. The numbers of s e c r e t o r y rosettes varied, d e p e n d i n g on the RBC type, and with some (goat, horse, calf, sheep and mouse) the p e r c e n t a g e s w e r e low (<5%) ( T a b l e i). However, these rosettes were m o r p h o l o g i c a l l y identical to those that formed in g r e a t e r n u m b e r s w i t h r a b b i t , rat a n d g u i n e a pig RBC. The diameters of the rosettes, a reflection of the amount of a g g l u t i n i n s e c r e t e d b y the l e u k o c y t e s , w e r e e q u i v a l e n t . The relative p e r c e n t a g e s of s e c r e t o r y rosettes formed in the presence of r a b b i t , rat, m o u s e and g u i n e a pig R B C are a p p r o x i m a t e l y p r o p o r t i o n a l to titers of induced fluid after injecting Lumbricus with these RBC (titers of 1,138, 767, 0.9 and 2.0, respectively) (15). In the case of induced fluid produced by injecting goat, h o r s e , c a l f a n d s h e e p RBC, a g g l u t i n i n s are p r o b a b l y p r e s e n t a g a i n s t these RBC, but there are too few leukocytes secreting them for the levels to be detected by c o n v e n t i o n a l a g g l u t i n a t i o n assays. The p e r c e n t a g e of E-type rosettes with those RBC's that had f o r m e d l o w e r n u m b e r s of s e c r e t o r y r o s e t t e s (rat, g u i n e a pig, mouse, sheep, calf, horse and goat), were also low, varying from 5.0-8.5% (Table i). However, E - r o s e t t e s with rabbit RBC (32.8%) were clearly in a d i f f e r e n t category, consistent with the relative d i f f e r e n c e s in numbers of s e c r e t o r y rosettes between rabbit and other RBC's. This suggests a possible r e l a t i o n s h i p between the RBC receptors of secretory and E-type rosettes; some leukocytes may not secrete an agglutinin but possess the
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IN VITRO AGGLUTININ PRODUCTION
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m o l e c u l e s in/on their cell membranes (for review, see Ii). L e u k o c y t e s from worms injected 24 hr earlier with rabbit RBC produced s i g n i f i c a n t l y fewer s e c r e t o r y rosettes when assayed with RBC from the rabbit used as the source of injected RBC (Fig. 3, T a b l e 5). Secretory rosette production with RBC from other rabbits was unaffected, and saline injection did not affect the levels of s e c r e t o r y rosettes. Prior RBC injection a p p a r e n t l y d e p l e t e d l e u k o c y t e s of a g g l u t i n i n s that could react with RBC from that rabbit. This d e p l e t i o n was not permanent, however, but was r e v e r s e d and a g g l u t i n i n s e c r e t i o n r e t u r n e d to n e a r l y n o r m a l l e v e l s by 24 hr (Fig. 3). C l e a r l y , this d e m o n s t r a t e s highly s p e c i f i c r e a c t i v i t y , b u t it is not yet p o s s i b l e to d e t e r m i n e whether the s p e c i f i c i t y resides in leukocyte r e c o g n i t i o n or in the a g g l u t i n i n s themselves, i.e., if the presence of any type of RBC s t i m u l a t e s the p r o d u c t i o n of a limited number of different agglutinins, only one of which can react with a given type of RBC, or if the l e u k o c y t e s r e c o g n i z e s p e c i f i c R B C a n d s e c r e t e a g g l u t i n i n s only for those RBC. In the c a s e of a l l o t y p i c RRBC, s p e c i f i c i t y of l e u k o c y t e r e c o g n i t i o n seems most likely, since only one set of leukocytes (those p r o d u c i n g a n t i - i n j e c t e d RRBC agglutinin) were d e p l e t e d of their agglutinin. A l t h o u g h previous studies have shown that 24 hrs after injecting worms with rabbit RBC, coelomic fluid a g g l u t i n i n levels were m a r k e d l y higher than in u n i n j e c t e d worms, these studies did not a t t e m p t to d e m o n s t r a t e a g g l u t i n i n - l e u k o c y t e s p e c i f i c i t y (15). However, coelomic fluid a g g l u t i n i n s which were produced after i n j e c t i n g RBC from d i f f e r e n t rabbits varied in their p h y s i o c h e m i c a l properties. In t h e c a s e of r o s e t t e f o r m a t i o n w i t h R B C f r o m d i f f e r e n t s p e c i e s , the s i t u a t i o n is l e s s c l e a r , s i n c e b o t h a g g l u t i n i n s p e c i f i c i t y and c r o s s - r e a c t i v i t y may occur. P e r c e n t a g e s of Erosettes in allogeneically stimulated worms were not s i g n i f i c a n t l y d i f f e r e n t for the two sources of RRBC except at the earlier assay times. A p p a r e n t l y d e p l e t i o n of i n t r a c e l l u l a r a g g l u t i n i n s did not m a r k e d l y affect the e x p r e s s i o n of surface molecules (agglutinins?) responsible for l e u k o c y t e - R B C binding of the E rosettes. A n u m b e r of sugars inhibited formation of s e c r e t o r y rosettes (Table 4), as did the p o l y s a c c h a r i d e m a n n a n and the g l y c o p r o t e i n s t h y r o g l o b u l i n and bovine s u b m a x i l l a r y mucin. P r e v i o u s studies have shown that rabbit RBC a g g l u t i n a t i o n by Lumbricus coelomic fluid was inhibited by only a few sugars, namely KDO, glucosamine, g a l a c t o s a m i n e and m a n n o s a m i n e (15,22). Of these, KDO was b e l i e v e d to be the only specific inhibitor, w h e r e a s the three amino sugars were thought to be i n h i b i t o r y by virtue of their positive charge (22). In the present study, KDO and the amino sugars inhibited secretory rosette formation; however, galactose, N-acetylgalactosamine, glucuronic acid and n-acetylneuraminic acid, which do not directly affect agglutination (22), inhibited s e c r e t o r y rosette formation. E r o s e t t e f o r m a t i o n w a s r e d u c e d b y s o m e i n h i b i t o r s , b u t not by o t h e r s , and in s o m e c a s e s the p e r c e n t a g e was a c t u a l l y h i g h e r (Table 4). This suggests that the site of action is not the same for all inhibitors, some acting on cell membrane receptors, some on the s e c r e t o r y a p p a r a t u s , and s o m e on b i n d i n g s i t e s of the a g g l u t i n i n s themselves. C y c l o h e x a m i d e did not affect s e c r e t o r y rosette formation,
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suggesting that agglutinin secretion in itself is not directly affected by inhibition of protein synthesis. More importantly, it suggests that the agglutinins are probably not synthesized during rosette formation but have been pre-formed and stored in the leukocytes, ready to be released after antigen (RBC) stimulation. This premise was implied in the previous observation, using allogeneic rabbit RBC, that restoration of a g g l u t i n i n s y n t h e s i s after d e p l e t i o n did not o c c u r rapidly. Approximately 24 hr were required for reconstitution of the full capacity to secrete a given agglutinin. The evidence suggests a greater degree of specificity than has generally been thought to occur at this phylogenetic level (23,24). Since earthworm hemagglutinins are also known to be bacterial agglutinins (20), such specificity may play a role in r e c o g n i t i o n and r e m o v a l of p a t h o g e n i c o r g a n i s m s and thus be essential to the earthworm's immunodefense system.
REFERENCES i. H a l l , J.L. and R o w l a n d s , D.T., H e t e r o g e n e i t y of l o b s t e r agglutinins. I. Purification and Physicochemical Characterization. Biochem. 13, 821, 1974. 2. Amirante, G.A. and Mazzalai, F.G. Synthesis and localization of hemoagglutinins in hemocytes of the cockroach Leucophaea maderae. Dev. Comp. Immunol. 2, 735, 1978. 3. Boerrigter-Barendsen, L.H., Van den Howven, D.S.P., Sminia, T. and Van der Knapp, W.P.W. Immunocytochemical demonstration of a humoral defense factor in blood cells (amoebocytes) of the pond snail Lymnaea sta~nalis. Cell Tiss. Res. 219, 291, 1981. 4. Vasta, G.R., Cheng, T.C. and Marchalonis, J.J. A lectin on the hemocyte membrane of the oyster (Creassostrea vir@inica). Cell. Immunol. 88, 475, 1984. 5. Cooper, E.L., Evolution of cellular immunity. In: Braun, W. and Ungar, J. (Eds.,) Non-Specific Factors Influencing Host Resistance, Karger, Basel 1973, p. II. 6. Toupin, J. and Lamoureux, G., 1976. Coelomocytes of earthworms: Phytohemagglutinin (PHA) responsiveness. In: Phylogeny of thymus and bone marrow bursa cells (R.K. Wright and E.L. Cooper, Eds.). North Holland, Amsterdam, pp. 19-26. 7. Chateaureynaud-Duprat, P. and Izoard, F. Compared study of i m m u n i t y b e t w e e n two genera of l u m b r i c i e n s : E i s e n i a and Lumbricus. In: Solomon, J.B. and Horton J.D. (Eds.), Developmental Immunobiology, Elsevier/North Holland, Amsterdam, New York 1977, p. 33. 8. Mohrig, W., Kauschke, E. and Ehlers, M. Rosette formation of the Coelomocytes of the earthworm Lumbricus terrestris with sheep erythrocytes. Dev. Comp. Immunol. 8, 471, 1984. 9. Anderson, R.S. Rosette formation by insect macrophages. Inhibition by cytochalasin B. Cell Immunol. 29, 331, 1973. 10. Wright, R.K. and Cooper, E.L. Tunicate leucocyte receptors a n d h e m o l y m p h lectins. In: Cohen E. (Ed.), R e c o g n i t i o n , Proteins, Receptors and Probes: Invertebrates, Alan R. Liss, New York 1984, p. 115.
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Stein, E.A. and Cooper, E.L. Agglutinins as receptor molecules: A phylogenetic approach. In: Cooper, E.L. and Brazier, M.A.B. (Eds.), Developmental Immunology: Clinical Problems and Aging, Academic Press, New York 1982, p. 85. 12. Hartree, E.F. Determination of protein. A modification of the Lowry method that gives a linear photometric response. Aqalyt. Biochem. 48, 422, 1972. 13 T o u p i n , J., Marks, D.H., Cooper, E.L. and L a m o u r e u x , G. Earthworm coelomocytes in vitro. In Vitro 13, 218, 1977. 14 Stein, E. and Cooper, E.L. The role of opsonins in phagocytosis by c o e l o m o c y t e s of the earthworm, L u m b r i c u s terrestris. Dev. Comp. Immunol. 5, 415, 1981. 15 Stein, E.A., Wojdani, A. and Cooper, E.L. Agglutinins in the earthworm, Lumbricus terrestris, naturally occurring and induced. Dev. Comp. Immunol 6, 407, 1982. 16 Young, C.W., Hendler, F.J. and Karnofsky, D.A. Synthesis of p r o t e i n for DNA replication and cleavage events in the sand dollar embryo. Exp. Cell Res. 58, 15, 1969. 17 Schneiderman, M.H., Dewey, W.C. and Highfield, D.P. Inhibition of DNA synthesis in synchronized Chinese hamster cells treated in G1 with cyclohexamide. Exp. Cell Res. 67, 147, 1971. 18 S c h e f l e r , W.C. S t a t i s t i c s for the B i o l o @ i c a l Sciences, Addison-Wesley, Reading 1979, p. 84. 19 Wojdani, A., Stein, E.A. and Cooper, E.L. Agglutinins and proteins in the earthworm, Lumbricus terrestris, before and a f t e r i n j e c t i o n of e r y t h r o c y t e s , c a r b o h y d r a t e s and other materials. Dev. Comp. Immunol. 6, 613, 1982. 20 Stein, E.A.,-Y0unai, S. and Cooper, E.L., In: E.L. Cooper, C. L a n g l e t and J. Bierne (Eds.) 3rd International Congress of Developmental and Comparative Immunolo~y~ Alan R. L1ss, New York, p. 133. 21 Stein, E.A., Avtalion, R.R. and Cooper, E.L. Coelomocytes of the earthworm, Lumbricus terrestris: Morphology and phagocytic properties. J. Morphol. 153, 467, 1977. 22 Stein, E.A. and Cooper, E.L. Carbohydrate and glycoprotein inhibitors of naturally occurring and induced agglutinins in the earthworm Lumbricus terrestris. Comp. Biochem. Physiol. D6B, 197, 1983. 23 Nisonoff, A., Hopper, J.E. and Spring, S.B. The Antibody Molecule, Academic Press, New York 1985, p. 264. 24 Klein, J. ~mmunology. The Science of Self-Nonself Discrimination, John Wiley'and Sons, New York 1982, p. 6. 25 Yeaton, R.W. Lectins of a North American Silkmoth (Hyalophora cecropia): Their Molecular Characterization and Developmental Biology , Ph.D. Th4sis~ ......University of Pennsylvania 1980. Received: June 1987 Accepted: October 1987
IN VITRO A G G L U T I N I N P R O D U C T I O N
BY EARTHWORM L E U K O C Y T E S
Elizabeth A. Stein and Edwin L. Cooper D e p a r t m e n t of Anatomy, School of Medicine, U n i v e r s i t y of California, Los Angeles, California
ABSTRACT
Leukocytes of the earthworm, Lumbricus terrestris, s e c r e t e a g g l u t i n i n s in vitro, as shown by measuring agglutinin titers of the culture medium and by observing secretory rosette formation by l e u k o c y t e s with erythrocytes. Leukocytes form the highest percentages of secretory rosettes with rabbit e r y t h r o c y t e s (RBC) and with other RBC species in the o r d e r : rat, g u i n e a pig, m o u s e , c a l f , s h e e p , h o r s e , goat. L e u k o c y t e s d i s p l a y e d a l l o t y p i c s p e c i f i c i t y by f o r m i n g rosettes s e l e c t i v e l y with e r y t h r o c y t e s from different individual rabbits. Eight sugars inhibited rosette formation, along with the p o l y s a c c h a r i d e mannan and the g l y c o p r o t e i n s t h y r o g l o b u l i n and bovine subm a x i l l a r y mucin. C y c l o h e x a m i d e did not affect rosette formation, s u g g e s t i n g that a g g l u t i n i n s may be p r e f o r m e d and stored in leukocytes prior to secretion. Leukocytes also formed E-type rosettes with erythrocytes, but a p p a r e n t l y utilized d i f f e r e n t receptors from those of s e c r e t o r y rosettes since they were not inhibited by the same sugars. INTRODUCTION
Hemocytes or l e u k o c y t e s of s e v e r a l i n v e r t e b r a t e s c o n t a i n agglutinins and presumably secrete them (1-4). However, agglutinin production has rarely been observed in vitro. R o s e t t e - f o r m i n g cells have been d e s c r i b e d in e a r t h w o r m s (5-8), insects (9) and tunicates (I0), but in most instances these have been E, type rosettes. Secretory rosettes have been d e s c r i b e d in L u m b r i c u s terrestris (7,11) and Styela clava (I0) and are formed 531
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IN VITRO AGGLUTININ PRODUCTION
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by a g g l u t i n i n - p r o d u c i n g cells. We show here that Lumbricus leukocytes can secrete a g g l u t i n i n s in vitro, by: I) direct assays of a g g l u t i n i n s in the c u l t u r e m e d i u m ; and 2) q u a n t i f y i n g s e c r e t o r y r o s e t t e - f o r m i n g cells. A s e c o n d c e l l type, the c h l o r a g o g e n cell, c o n t a i n s agglutinins, but because of their fragility have not been cultured in vitro. Lumbricus leukocytes exhibit s p e c i f i c i t y in their reactions with erythrocytes; the p e r c e n t a g e s of s e c r e t o r y and E-type rosettes vary, d e p e n d i n g on the e r y t h r o c y t e species. It is of i n t e r e s t t h a t l e u k o c y t e s a l s o r e c o g n i z e a l l o t y p i c differences, forming secretory rosettes specifically with erythrocytes from d i f f e r e n t i n d i v i d u a l s of the same s p e c i e s . Assays using secretory rosettes now provide a tool for analyzing the p r o d u c t i o n of i n v e r t e b r a t e a g g l u t i n i n s and for a s s a y i n g humoral immune responses at the c e l l u l a r level.
M A T E R I A L S AND METHODS
Earthworms: Adult earthworms (Lumbricus terrestris) were purchased from Golden West Cricket Co., Paramount, CA and m a i n t a i n e d at 1 5 ° C in B u s s B e d d i n g ( B u s s M a n u f a c t u r i n g Co., Lanark, IL) with supplemental feedings of dry baby cereal. Agglutinin content in Iysates of leukocytes: Agglutinin levels were measured {tom two cell sources, l e u k o c y t e s and c h l o r a g o g e n cells. Leukocytes, c o n t a i n e d in coelomic fluid, were c o l l e c t e d by inserting a sharpened Pasteur pipette into the coelomic cavity posterior to the c l i t e l l u m and allowing it to fill with coelomic fluid by intra-coelomic pressure. The fluid was c e n t r i f u g e d at I00 g, s u p e r n a t a n t removed and l e u k o c y t e s washed three timesli~0 normal (0.85%) saline. Cells were counted, a d j u s t e d to 1 x cells/ml and l y s e d by a s o n i c d i s r u p t e r . The l y s a t e was c e n t r i f u g e d at 10,000g for 30 min to remove particulate matter and the supernatant retained for assays. A g ~ l u t i n i n content in Iysates of c h l o r a g o g e n cells: C h l o r a g o g e n c e l l s w e r e c o l l e c t e d by a n e s t h e t i z i n g w o r m s for 1 hr in 5% ethanol in water (v/v) then cutting them open longitudinally. Chloragogen cells, which invest the outer surface of the digestive t r a c t , w e r e g e n t l y s c r a p e d off w i t h a s c a l p e l and suspended in 0.85% saline. Since cells were disrupted, it was i m p o s s i b l e to p e r f o r m c e l l c o u n t s ; h o w e v e r , s u s p e n s i o n s w e r e checked microscopically to i n s u r e that t h e r e was n e g l i g i b l e c o n t a m i n a t i o n by leukocytes. Cell samples were collected from 12 worms, as three samples from four worms. The cell suspension was lysed by sonic d i s r u p t i o n and c e n t r i f u g e d as for leukocytes to remove particulate matter. Leukocyte and chloragogen cell lysates were assayed for a g g l u t i n a t i o n titers using rabbit erythrocytes (RRBC) (see below) and for protein c o n c e n t r a t i o n s by a modified Lowry procedure (12). ~eqkocyte collection for in vitro cell cultures: Earthworm coelomic fluid, c o n t a i n i n g leukocytes, was removed a s e p t i c a l l y as p r e v i o u s l y d e s c r i b e d (13). Briefly, worms were a n e s t h e t i z e d as d e s c r i b e d a b o v e , then g e n t l y , but r a p i d l y s c r u b b e d w i t h 95% ethanol to reduce external contamination, and rinsed in sterile 0.85% saline. Worms were then placed in sterile petri dishes and cut o p e n l o n g i t u d i n a l l y on the d o r s a l s u r f a c e to e x p o s e the
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IN VITRO AGGLUTININ PRODUCTION
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coelomic cavity. C o e l o m i c fluid and l e u k o c y t e s were washed out with sterile saline, c o l l e c t e d by sterile Pasteur pipettes and c e n t r i f u g e d for 5 min at 150 g. The supernatant was removed and cells w a s h e d twice in saline. Assays of in vitro ag@lutinin production: Leukocytes were r e s u s p e n d e d in a modified L i e b o v i t z - 1 5 (L-15) medium consisting of: L-15 medium c o n t a i n i n g L-glutamine, 60% (v/v); Lumbricus b a l a n c e d salt solution (LBSS) (14), 40%: gentamycin, 5 mg/100 ml. Fetal calf serum used p r e v i o u s l y in e a r t h w o r m culture media (33), was not added, since it inhibited coelomic fluid a g g l u t i n a t i o n of erythrocytes, and did not increase leukocyte v i a b i l i t y in our assays. The pH was a d j u s t e d to 7.2 and the medium sterilized by filtration. L e u k o c y t e d e n s i t y was adjusted to 1 x i0 cells/ml and the s u s p e n s i o n added as 0.2 ml a l i q u o t s to wells of sterile, 96-well, flat-bottom culture plates (Costar Plastics, M~A Bioproducts, Los Angeles, CA). Cultures were m a i n t a i n e d at 15 C and monitored periodically for contamination. At specific intervals, contents of the wells were removed, centrifuged to remove cells and p a r t i c u l a t e matter, and the supernatant assayed for a g g l u t i n i n titers. In vitro a ~ l u t i n i n p r o d u c t i o n in the presence of erythrocytes: A separate set of experiments was designed to determine if agglutinin i n d u c t i o n w o u l d o c c u r in v i t r o , as we p r e v i o u s l y demonstrated in vivo (15). ~eukocyte concentrations were adjusted before plating to 2 x I0v cells/ml and an equal volume of 0 . 1 2 5 % (v/v) R R B C s u s p e n s i o n (see b e l o w ) was a d d e d . The suspension of mixed leukocytes and RRBC was plated as before in sterile m i c r o t i t e r plates at 0.2 ml/well. Fluid was removed from the wells at specific intervals and titers of the supernatant determined. T i t e r s of s e c r e t e d a g g l u t i n i n : F i f t y m i c r o l i t e r s of c u l t u r e supernatant, either from wells c o n t a i n i n g leukocytes alone or l e u k o c y t e s plus RRBC, were added to wells of U-type m i c r o t i t e r plates. The fluid was serially d i l u t e d with p h o s p h a t e - b u f f e r e d saline (PBS) and 25 1 of a 2% (v/v) RRBC suspension in PBS added to each well. Plates were shaken gently for several minutes, then a l l o w e d to stand for 1 hr. Titers were read and recorded as the r e c i p r o c a l of the highest d i l u t i o n showing agglutination. Erythrocytes: R a b b i t b l o o d was c o l l e c t e d in h e p a r i n f r o m the marginal ear vein and washed three times in 0.85% saline. The buffy coat was removed and e r y t h r o c y t e s (RRBC) r e - s u s p e n d e d at a d e n s i t y of 2% (v/v) in LBSS that had been adjusted with NaCI to 300mOsM (LBSS-300). For some experiments, we used a panel of d i f f e r e n t erythrocytes. Guinea pig, calf and goat blood were purchased from Colorado Serum Co., Denver, CO. Sheep and horse b l o o d were purchased from Mission L a b o r a t o r y Supply, Rosemead, CA. Rat and mouse blood were kindly supplied by Dr. Yoko Mullen, Dental Research Institute, University of California at Los Angeles. All types of blood were p r o c e s s e d as for RRBC. Isolation and p r e p a r a t i o n of leukocytes for rosetting: Coelomic f l u i d w a s c o l l e c t e d b y i n s e r t i n g a P a s t e u r p i p e t t e into the c o e l o m i c cavity, as d e s c r i b e d above. The fluid was then mixed gently with cold 0.85% saline and c e n t r i f u g e d at 150 g for i0 min. The s u p e r n a t a n t was removed, cells washed once more and r e s u s p e n d e d in 0.2 ml saline, then mixed with 0.8 ml LBSS-300. By w a s h i n g l e u k o c y t e s i n i t i a l l y in s a l i n e , then r e s u s p e n d i n g l a t e r in L B S S - 3 0 0 , c l u m p i n g was r e d u c e d and l e s s c e l l d a m a g e
534
IN VITRO AGGLUTININ PRODUCTION
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o c c u r r e d during resuspension. Cell d e n s i t y was adjusted to I0 6 c e l l s / m l in LBSS-300. O n e - t e n t h ml a l i q u o t s were placed in small tubes and kept at 4°C. L e u k o c y t e s from each worm were c o l l e c t e d and p r o c e s s e d s e p a r a t e l y for all assays. Identification of rosettes: Two percent s u s p e n s i o n s of RBC in L B S S - 3 0 0 w e r e a d d e d as 0.i m l ~ l i q u o t s to an e q u a l v o l u m e of leukocytes in LBSS-300 at i 0 v cells/ml. Leukocytes and e r y t h r o c y t e s were mixed briefly, c e n t r i f u g e d at i00 g for 5 min, then kept at 4vC (see below) for specific time periods. Just before rosettes were to be counted, 0.I ml of a solution of 0.1% crystal violet in LBSS-300 was added to the cell s u s p e n s i o n to stain cell nuclei. Tubes were shaken g e n t l y to r e - s u s p e n d the p e l l e t a n d a s m a l l a m o u n t of the c e l l s u s p e n s i o n p l a c e d in a h e m o c y t o m e t e r chamber. One hundred cells were counted for each sample at each time-point. Fourteen to sixteen worms were used for e a c h set of a s s a y s , each worm providing cells for all time-points. Cells were scored either as s e c r e t o r y rosettes, E-rosettes or non-rosetting cells. Secretory rosettes are leukocytes surrounded by two or more layers of adhering erythrocytes. E-rosettes are leukocytes w i t h f o u r or m o r e erythrocytes adhering to the c e l l s u r f a c e as a s i n g l e l a y e r . N o n - r o s e t t i n g cells c o n s i s t e d of those l e u k o c y t e s with fewer than four a d h e r i n g e r y t h r o c y t e s . Rosette formation was measured with each of the d i f f e r e n t types of erythrocytes: rabbit, rat, mouse, guinea pig, calf, goat and horse. Carbohydrate and g l y c o p r o t e i n inhibition of rosette formation: Sugars and glycoproteins~ as s o l u t i o n s in PBS, w e r e a d d e d to suspensions of Lumbricus leukocytes just before mixing the leukocytes with rabbit RBC. Cultures were scored at 4 hr incubation for s e c r e t o r y and E-type r o s e t t e s as d e s c r i b e d above. The D isomers of glucose, galactose, mannose, glucosamine, galactosamine, mannosamine, N-acetylglucosamine, N-acetylgalactosamine, N-acetylmannosamine, glucuronic acid, -methylmannoside, a n d a r a b i n o s e w e r e used. We a l s o t e s t e d L-fucose, N - a c e t y l n e u r a m i n i c acid and 2 - k e t o - 3 - d e o x y o c t o n a t e (all sugars at 50 mM), the p o l y s a c c h a r i d e mannan (2 mg/ml) and the glycoproteins bovine submaxillary mucin (0.2 mg/ml), and thyroglobulin (2 m g / m l ) . The pH of all s u g a r s o l u t i o n s w a s a d j u s t e d to 7.2. C o n c e n t r a t i o n s were used that had been shown p r e v i o u s l y to inhibit RRBC a g g l u t i n a t i o n (15). Temperature effects on rosette formation: The effects of temperature on r o s e t t e f o r m a t i o n w e r e d e t e r m i n e d in a set of parallel assays p e r f o r m e d at 5°C and 15°C. L e u k o c y t e suspensions were p r e p a r e d and mixed with 2% RRBC s u s p e n s i o n s as d e s c r i b e d above. D u p l i c a t e c u l t u r e s were p r e p a r e d for each time point, one set k e p t at 5 C a n d the o t h e r at 15°C. At s p e c i f i e d times, c u l t u r e s were e x a m i n e d and the s e c r e t o r y and E - r o s e t t e s counted. Rosette formation by l e u k o c y t e s from injected worms: Worms were injected with 0.10-0.15 ml of a 5% s u s p e n s i o n of RRBC in 0.85% saline (15). RRBC were used from two d i f f e r e n t sources, rabbit A (RRBC A) and rabbit M (RRBC M). T w e n t y - f o u r hr later, coelomic fluid was c o l l e c t e d and leukocytes ~rocessed, as d e s c r i b e d above, for assays of rosette formation. Aliquots from each worm were divided into two s e t s ; one set to be a s s a y e d for r o s e t t e formation with erythrocytes from the same rabbit that had supplied the injected RBC, and a second set to be assayed with RBC from a rabbit d i f f e r e n t from the one used for injection. The
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converse experiment was also performed, injecting worms with RRBC from the second source and assaying with both types of RRBC. Cyclohexamide: To determine if suppression of protein synthesis would affect rosette formation, leukocytes and RRBC were cultured in LBSS-300 containing I0 ~ g/ml cyclohexamide (16, 17). Parallel cultures were tested by counting 200 cells, stained with 0.1% trypan blue dye, to determine cell viability in the presence and absence of cyclohexamide. Statistics: Significance levels were determined by the Student's T. test (18). RESULTS Agglutinin levels in leukocytes and chlorago~en cells: Leukocyt~ lysates were assayed and calculated for agglutinin levels per 10cells and per mg protein. Chloragogen cells, since cell counts were not possible, were calculated only as agglutinin units per mg p r o t e i n . L e u k o c y t e s and c h l o r a g o g e n c e l l s b o t h c o n t a i n e d hemagglutinins (Table I). Leukocytes contained 7specific titers of 6.7 units per mg protein and 12.7 units per i0 cells, whereas chloragogen cells contained 41.7 units per mg protein. In vitro a~glutinin production by Lumbricus leukocytes: Leukocytes cultured in nutrient medium r e l e a s e d low levels of hemagglutinins, as shown by measurement of agglutination titers of the supernatant medium. Highest agglutinin levels occurred at 24 and 48 hr, w i t h m e a n t i t e r s of i0.i and 10.2 r e s p e c t i v e l y (Table 2). In those cultures to which rabbit erythrocytes had b e e n a d d e d , a g g l u t i n i n t i t e r s w e r e s l i g h t l y l o w e r than those cultures with leukocytes alone (Table 2). Rosette formation with erythrocytes: Earthworm leukocytes formed b o t h s e c r e t o r y and E - t y p e r o s e t t e s w i t h e r y t h r o c y t e s of all species tested (Table 3). However, the highest percentages of secretory rosettes occurred using RRBC; at 6 hr, 26.7% of the leukocytes formed secretory rosettes (Fig. i). Percentages of E-type rosettes with RRBC ranged from a high of 34.9% at 30 min to 27.1% at 2 hr and rose again slightly at 4 hr to 32.8% (Fig. 2). Using other erythrocytes, the p e r c e n t a g e s of r o s e t t e s varied. With sheep erythrocytes (SRBC), l e u k o c y t e s f o r m e d a maximum of 2.7% secretory rosettes at 2 to 8 hr incubation (Fig. i), and 15.6% E - t y p e r o s e t t e s at 2 hr (Fig. 2). Using other erythrocytes, at 4 hr incubation, secretory rosettes ranged from 0.7% (goat RBC) to 8.5% (rat RBC), and E-type rosettes varied from 8.5% to 15% (Table 3). Carbohydrate and glycoprotein inhibition of rosette formation: Of the 15 sugars tested, 8 inhibited secretory rosette formation to a significant ( P < 0 . 0 5 ) d e g r e e ( T a b l e 4). The p o l y s a c c h a r i d e mannan and the glycoproteins thyroglobulin and bovine submaxillary mucin were also inhibitory. Conversely, E rosette formation was not reduced but instead increased with 8 of the substances, and remained unchanged with the other 3 (Table 4). Amino sugars (glucosamine, galactosamine and mannosamine) were consistently inhibitory, but the acetylated form was inhibitory only for N-acetylgalactosamine. Effects of temperature on rosette formation: Leukocytes incubated at 15-C wi~h RRBC formed secretory rosettes somewhat faster than those at 5-C, with maximum numbers of rosettes occurring at 2-4
536
IN VITRO AGGLUTININ PRODUCTION
TABLE
Agglutinin
Cell
1
L e v e l s of L u m b r i c u s L e u k o c y t e s and C h l o r a g o g e n Cells
type
Per
i0
7
cells
12.7 + 5.5
Chloragogen cells
.
(26)
.
.
.
1.9 + 0.5 .
TABLE
Hours in culture
of
a
5.0
12
+ 0.8
(6)
7.0 + 0.7
(6)
(13)
41.7
(15)
of worms
by L u m b r i c u s
T i t e r s a of p a i r e d l e u k o c y t e b c u l t u r e s , in p r e s e n c e and a b s e n c e of r a b b i t RBC Leukocytes only
6
RBC. Number
6.7
2
In vitro A g g l u t i n i n Leukocytes
A g g l u t i n i n tit~rs of l e u k o c y t e c u l t u r e media
(13)
.
T i t e r s m e a s u r e d by a g g l u t i n a t i o n of r a b b i t S a m p l e mean + s t a n d a r d e r r o r of the mean. given in ( )?
Production
T i t e r a per mg. p r o t e i n (Specific titer)
Protein concentration (mg)
Titer a
Leukocytes
Vol. 12, No. 3
L e u k o c y t e s plus r a b b i t RBC c
24
I0.i
+ 1.5
(20)
4.3
+ 0.6
(9)
3.8 + i.i
(9)
48
10.2 + 2.3
(20)
9.8
+
1.7
(9)
6.7
+
1.5
(9)
(18)
4.5 + 0.8
(9)
2.9
+ 0.6
(9)
72 a
b c
8.4
+ 1.8
Mean + s t a n d a r d e r r o r of the mean. the r ~ c i p r o c a l of the last d i l u t i o n N u m b e r of w o r m s given in ( ). 1 x 106 l e u k o c y t e s
/ml.
0.125%
v/v.
rabbit
RBC,
T i t e r s e x p r e s s e d as showing agglutination.
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IN VITRO AGGLUTININ PRODUCTION
TABLE
537
3
P e r c e n t of L u m b r i c u s L e u k o c y t e s that form S e c r e t o r y and E - r o s e t t e s w i t h D i f f e r e n t T y p e s of E r y t h r o c y t e s (RBC)
Percent
rosettes a
Type of RBC
Secretory M e a n + SEM
E-type M e a n + SEM
rabbit
23.5
+ 1.7
32.8
+ 3.5
(16)
8.5 + 1.0
14.1
+ 1.6
(8)
5.2
15.0
+ 2.7
(5)
8.5 + 1.2
(8)
rat guinea
pig
+ 1.3
N u m b e r of worms
mouse
2.4 + 0.9
sheep
2.7 + I.I
11.2
+ 1.5
(8)
calf
3.2 + 1.3
9.4
+ 1.2
(5)
horse
1.4
+ 0.5
9.6
+ 1.2
(I0)
goat
0.7 + 0.6
14.6
+ 2.3
(i0)
a
After
4 hr.
incubation
at 4°C.
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IN VITRO AGGLUTININ PRODUCTION
TABLE
Vol. 12, No. 3
4
I n h i b i t i o n of R o s e t t e F o r m a t i o n by C a r b o h y d r a t e s and G l y c o p r o t e i n s
Percentage Substance a
in N u m b e r s RRBC
Decrease b of S e c r e t o r y
Rosettes
Percent
Increase b
in N u m b e r s RRBC
of
Rosettes
Galatose
54.8
+ 5.5
+26.4
+ 6.2
Galactosamine
54.2
+
+23.4
+
N-acetylgalactosamine
33.1
+ 5.4
Glucuronic
22.3
+
4.9
+16.0
+
Glucosamine
45.2
+ 6.9
+30.7
+ 7.5
Mannosamine
57.6
+
6.2
+29.0
+
55.6
+ 4.9
+32.8
+ 7.4
2-keto-3-deoxyoctonate
52.7
+ 5.1
0
Mannan
22.3
+ 4.8
0
Thyroglobulin
52.4
+
5.7
+46.9
+
26.9
+ 5.2
+41.6
+ 8.4
acid
N-acetylneuraminic
Bovine
submaxillary
acid
mucin
6.1
E
3.2
0 4.4
5.7
7.9
O n l y those s u b s t a n c e s s i g n i f i c a n t l y (P<0.05) i n h i b i t o r y are listed. M o n o s a c c h a r i d e s w e r e used at 50 mM, m a n n a n and t h y r o g l o b u l i n at 2 mg/ml, b o v i n e s u b m a x i l l a r y mucin at 0.2 mg/ml. b
P e r c e n t change, a f t e r 4 hr of c u l t u r e , from n u m b e r s of r o s e t t e s formed in a b s e n c e of i n h i b i t o r , + s t a n d a r d error of mean.
4
culture
Hrs in A
33.4+10.0 3 4 . 3 T 5.7
(9) (8)
Mean
(9) (8)
(I0) (9)
+ standard
29.7+6.8 28.0T4.9
20.4+7.3 30.5T8.1
RRBC M
rosettes b with:
33.2+8.8 (I0) 29.8TII.8 (9)
RRBC
Secretory
24 hrs previously
4 24
24
5
with
deviation.
25.3+5.6 19.0~4.0
25.5+6.8 18.7T7.8
RRBC
A
(9) (8)
(I0) (9)
28.8+3.8 19.8~3.4
22.4+7.8 21.4+5.8
RRBC M
E rosettes b with:
For,nation by Leukocytes from Worms Previously Injected Saline or with Erythrocytes from Rabbit M
bpercent of total leukocytes. Number of worms in ( ).
aInjected
Saline
RRBC M
with
Injected
Rosette
TABLE
(9) (9)
(I0) (9)
%a %0 ~D
0 Z
0
Z
Z
%0
Z o
< o
540
IN VITRO AGGLUTININ PRODUCTION
Vol. 12, No. 3
26 26 24
~22 h-
~ m I1:
•
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Fig.
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S e c r e t o r y r o s e t t e s formed with rabbit and sheep RBC. Lumbricus leukocytes forming secretory rosettes with r a b b i t and sheep RBC are given as a percent of the total number of leukocytes. X ~ S.D.
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Fig.
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E-type r o s e t t e s formed with r a b b i t and sheep RBC. The numbers of leukocytes forming E - r o s e t t e s with rabbit and sheep RBC are given as a percent of the total number of leukocytes. X + S.D.
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IN VITRO AGGLUTININ PRODUCTION
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26 24 22 20
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~4 HOURS SECRETORY ROSETTES WITH RRBC A AND M (WORMS PREVIOUSLY INJECTED WITH RRBC A)
Fig.
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Secretory r o s e t t e s formed w i t h r a b b i t RBC of d i f f e r e n t a l l o t y p e s (RBC A and RBC M). L e u k o c y t e s were taken from w o r m s p r e v i o u s l y i n j e c t e d w i t h RBC from r a b b i t A. X + S.D.
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14 HOURS E-TYPE ROSETTES WITH RRBC A AND M (WORMS PREVIOUSLY INJECTED WITH RRBC A)
Fig.
4.
E-type rosettes f o r m e d w i t h r a b b i t R B C of d i f f e r e n t a l l o t y p e s (RBC A and RBC M). L e u k o c y t e s were taken from w o r m s p r e v i o u s l y i n j e c t e d w i t h RBC from r a b b i t A. X + S.D.
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hr and 4-6 hr, r e s p e c t i v e l y (not illustrated). However, the maximum p e r c e n t a g e s of s e c r e t o r y rosettes were slightly higher for cultures m a i n t a i n e d at 5°C (33%) than those at 15UC (31%). In addition, l e u k o c y t e v i a b i l i t y at 24 hr was slightly higher for cultures m a i n t a i n e d at 5°C (79.5 + 6.4, mean + S.D.) than those at 15°C (72.2 + 8.4). --Rosette formation by leukocytes from injected earthworms. D i s t i n c t i o n b e t w e e n e r y t h r o c y t e a l l o t y p e s : L e u k o c y t e s "{rom worms p r e v i o u s l y injected with RRBC from rabbit A produced d e c r e a s e d numbers of s e c r e t o r y rosettes when tested with e r y t h r o c y t e s from rabbit A (Fig. 3). The p e r c e n t a g e s of RRBC-A s e c r e t o r y rosettes, formed with leukocytes from RRBC-A-injected worms, were approximately 50% lower than normal (Fig. i), and were s i g n i f i c a n t l y lower (p < 0.01) for all time-points from 30 min t h r o u g h 6 hr. At 8 hr--of c u l t u r e , the n u m b e r s of s e c r e t o r y rosettes increased slightly, and at 24 hr the percentage was back to n e a r l y normal levels. Parallel cultures of leukocytes from worms injected with RRBC-A, tested with e r y t h r o c y t e s from rabbit M, produced normal levels of s e c r e t o r y rosettes (approximately 24% at 4 hrs) (Fig. 3). P e r c e n t a g e s of E-type rosettes formed with RRBC-A by leukocytes from worms injected with R R B C - A were somewhat lower than normal but s i g n i f i c a n t l y so only at 30 min; those tested with RRBC-M were at normal levels (Fig. 4). The converse experiment, injecting worms with RRBC-M and m e a s u r i n g rosette formation with RRBC-A and RRBC-M, resulted in a similar r e d u c t i o n of secretory rosettes when tested with RRBC M (Table 5). E rosettes were not s i g n i f i c a n t l y affected. L e u k o c y t e s from worms p r e v i o u s l y injected with saline formed secretory and E rosettes at normal levels when a s s a y e d with both RRBC A and RRBC M (Table 5). Effect of c y c l o h e x a m i d e on rosette fo[mation: The a d d i t i o n of c y c l o h e x a m i d e to the culture m e d i u m did not affect the levels of secretory rosettes w i t h i n the 24-hr period (Fig. 5). Levels of E - r o s e t t e s were slightly, but not significantly, lower in the c y c l o h e x a m i d e - c o n t a i n i n g c u l t u r e s than in regular medium (Fig. 6). V i a b i l i t y of b o t h control and c y c l o h e x a m i d e - t r e a t e d leukocytes decreased e q u a l l y w i t h time. O v e r the 2 4 - h r c u l t u r e p e r i o d , v i a b i l i t y of b o t h s e t s of l e u k o c y t e s was r e d u c e d by a p p r o x i m a t e l y 10%. V a r i a t i o n in v i a b i l i t y was p r i m a r i l y a factor of the p a r t i c u l a r worm from which the leukocytes were harvested, rather than the presence of cyclohexamide. Initial v i a b i l i t y for both sets of cultures varied from 83-97% (X + S.D. = 88.3 + 6.8), d e c r e a s i n g to 7 5 - 8 8 % a t 24 hr (77.4 + 8.5 for c u l t u r e ~ w i t h c y c l o h e x a m i d e , 79.1 + 8.5 for cultures in LBSS alone).
DISCUSSION The coelomic fluid of L u m b r i c u s contains n a t u r a l l y o c c u r r i n g a g g l u t i n i n s against b a c t e r i a and v e r t e b r a t e erythrocytes, and e l e v a t e d a g g l u t i n i n l e v e l s may be i n d u c e d b y i n j e c t i n g t h e s e antigens (15,19). The a g g l u t i n i n s r e a c t w i t h a n u m b e r of v e r t e b r a t e RBC, y i e l d i n g highest titers with rabbit RBC (15). Bacterial a g g l u t i n i n s react with both G r a m - p o s i t i v e and Gramnegative bacteria, and a v a i l a b l e evidence suggests that some of them are identical to the h e m a g g l u t i n i n s (20).
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IN VITRO AGGLUTININ PRODUCTION
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30
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5.
Secretory rosettes formed with rabbit RBC in the p r e s e n c e and a b s e n c e of c y c l o h e x a m i d e . Cyclohexamide, I0 g/ml, was continuously present in o n e set of cultures. X + S.D.
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Fig.
6.
E - t y p e r o s e t t e s formed w i t h r a b b i t RBC in the p r e s e n c e a n d a b s e n c e of c y c l o h e x a m i d e . Cyclohexamide, i0 g/ml, was c o n t i n u o u s l y p r e s e n t in one set of c u l t u r e s . X + S.D.
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The source of a g g l u t i n i n s appears to be twofold - l e u k o c y t e s and c h l o r a g o g e n cells. Free c h l o r a g o g e n cells are fragile and not c o m m o n l y found in coelomic fluid, but their cell contents (granules and vesicles) are frequently visible in coelomic fluid or c o n t a i n e d w i t h i n p h a g o c y t i c v a c u o l e s of l e u k o c y t e s (21). Although both types of cells contain agglutinins, c h l o r a g o g e n cells are higher on a per mg protein basis (Table i); however, b e c a u s e of t h e i r f r a g i l i t y , t h e y c a n n o t be e a s i l y c u l t u r e d . L e u k o c y t e s of s e v e r a l o t h e r i n v e r t e b r a t e s h a v e b e e n s h o w n to c o n t a i n agglutinins, including the lobster, Homarus a m e r i c a n u s (I), the cockroach, Leucophaea maderae (2), the silkmoth, Hyalophora cepropia (25), the snail, Lymnaea stagnalis (3) and the oyster, Crassostrea virginica (4). Lumbricus leukocytes secrete small but measurable amounts of a g g l u t i n i n when cultured in vitro. Levels in the culture medium w e r e low ( m a x i m u m t i t e r s of 10.2), w h e n c o m p a r e d to the h i g h titers (up to 1,024) c o m m o n l y found in coelomic fluid from worms that had been previously immunized. Several factors may c o n t r i b u t e to this 100-fold difference: less than optimum culture conditions for l e u k o c y t e s ; lower cell concentration t h a n in coelomic fluid; lack of a c c e s s o r y factors that might be required to stimulate leukocytes to synthesize a n d / o r secrete agglutinins. An attempt to correct this last problem by adding an inducing agent, rabbit RBC, to the c u l t u r e s was not successful. However, since these RBC were visibly a g g l u t i n a t e d after a few hours, they may h a v e a b s o r b e d and r e m o v e d f r o m the f l u i d s o m e of the agglutinins that would otherwise have been available for m e a s u r e m e n t in the assays. R o s e t t e formation is a more sensitive method for d e m o n s t r a t ing a g g l u t i n i n secretion than a g g l u t i n a t i o n titers of culture media. U s i n g r o s e t t i n g , we f o u n d t h a t L u m b r i c u s l e u k c o y t e s secreted a g g l u t i n i n s that reacted with all of the erythrocyte types. The numbers of s e c r e t o r y rosettes varied, d e p e n d i n g on the RBC type, and with some (goat, horse, calf, sheep and mouse) the p e r c e n t a g e s w e r e low (<5%) ( T a b l e i). However, these rosettes were m o r p h o l o g i c a l l y identical to those that formed in g r e a t e r n u m b e r s w i t h r a b b i t , rat a n d g u i n e a pig RBC. The diameters of the rosettes, a reflection of the amount of a g g l u t i n i n s e c r e t e d b y the l e u k o c y t e s , w e r e e q u i v a l e n t . The relative p e r c e n t a g e s of s e c r e t o r y rosettes formed in the presence of r a b b i t , rat, m o u s e and g u i n e a pig R B C are a p p r o x i m a t e l y p r o p o r t i o n a l to titers of induced fluid after injecting Lumbricus with these RBC (titers of 1,138, 767, 0.9 and 2.0, respectively) (15). In the case of induced fluid produced by injecting goat, h o r s e , c a l f a n d s h e e p RBC, a g g l u t i n i n s are p r o b a b l y p r e s e n t a g a i n s t these RBC, but there are too few leukocytes secreting them for the levels to be detected by c o n v e n t i o n a l a g g l u t i n a t i o n assays. The p e r c e n t a g e of E-type rosettes with those RBC's that had f o r m e d l o w e r n u m b e r s of s e c r e t o r y r o s e t t e s (rat, g u i n e a pig, mouse, sheep, calf, horse and goat), were also low, varying from 5.0-8.5% (Table i). However, E - r o s e t t e s with rabbit RBC (32.8%) were clearly in a d i f f e r e n t category, consistent with the relative d i f f e r e n c e s in numbers of s e c r e t o r y rosettes between rabbit and other RBC's. This suggests a possible r e l a t i o n s h i p between the RBC receptors of secretory and E-type rosettes; some leukocytes may not secrete an agglutinin but possess the
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IN VITRO AGGLUTININ PRODUCTION
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m o l e c u l e s in/on their cell membranes (for review, see Ii). L e u k o c y t e s from worms injected 24 hr earlier with rabbit RBC produced s i g n i f i c a n t l y fewer s e c r e t o r y rosettes when assayed with RBC from the rabbit used as the source of injected RBC (Fig. 3, T a b l e 5). Secretory rosette production with RBC from other rabbits was unaffected, and saline injection did not affect the levels of s e c r e t o r y rosettes. Prior RBC injection a p p a r e n t l y d e p l e t e d l e u k o c y t e s of a g g l u t i n i n s that could react with RBC from that rabbit. This d e p l e t i o n was not permanent, however, but was r e v e r s e d and a g g l u t i n i n s e c r e t i o n r e t u r n e d to n e a r l y n o r m a l l e v e l s by 24 hr (Fig. 3). C l e a r l y , this d e m o n s t r a t e s highly s p e c i f i c r e a c t i v i t y , b u t it is not yet p o s s i b l e to d e t e r m i n e whether the s p e c i f i c i t y resides in leukocyte r e c o g n i t i o n or in the a g g l u t i n i n s themselves, i.e., if the presence of any type of RBC s t i m u l a t e s the p r o d u c t i o n of a limited number of different agglutinins, only one of which can react with a given type of RBC, or if the l e u k o c y t e s r e c o g n i z e s p e c i f i c R B C a n d s e c r e t e a g g l u t i n i n s only for those RBC. In the c a s e of a l l o t y p i c RRBC, s p e c i f i c i t y of l e u k o c y t e r e c o g n i t i o n seems most likely, since only one set of leukocytes (those p r o d u c i n g a n t i - i n j e c t e d RRBC agglutinin) were d e p l e t e d of their agglutinin. A l t h o u g h previous studies have shown that 24 hrs after injecting worms with rabbit RBC, coelomic fluid a g g l u t i n i n levels were m a r k e d l y higher than in u n i n j e c t e d worms, these studies did not a t t e m p t to d e m o n s t r a t e a g g l u t i n i n - l e u k o c y t e s p e c i f i c i t y (15). However, coelomic fluid a g g l u t i n i n s which were produced after i n j e c t i n g RBC from d i f f e r e n t rabbits varied in their p h y s i o c h e m i c a l properties. In t h e c a s e of r o s e t t e f o r m a t i o n w i t h R B C f r o m d i f f e r e n t s p e c i e s , the s i t u a t i o n is l e s s c l e a r , s i n c e b o t h a g g l u t i n i n s p e c i f i c i t y and c r o s s - r e a c t i v i t y may occur. P e r c e n t a g e s of Erosettes in allogeneically stimulated worms were not s i g n i f i c a n t l y d i f f e r e n t for the two sources of RRBC except at the earlier assay times. A p p a r e n t l y d e p l e t i o n of i n t r a c e l l u l a r a g g l u t i n i n s did not m a r k e d l y affect the e x p r e s s i o n of surface molecules (agglutinins?) responsible for l e u k o c y t e - R B C binding of the E rosettes. A n u m b e r of sugars inhibited formation of s e c r e t o r y rosettes (Table 4), as did the p o l y s a c c h a r i d e m a n n a n and the g l y c o p r o t e i n s t h y r o g l o b u l i n and bovine s u b m a x i l l a r y mucin. P r e v i o u s studies have shown that rabbit RBC a g g l u t i n a t i o n by Lumbricus coelomic fluid was inhibited by only a few sugars, namely KDO, glucosamine, g a l a c t o s a m i n e and m a n n o s a m i n e (15,22). Of these, KDO was b e l i e v e d to be the only specific inhibitor, w h e r e a s the three amino sugars were thought to be i n h i b i t o r y by virtue of their positive charge (22). In the present study, KDO and the amino sugars inhibited secretory rosette formation; however, galactose, N-acetylgalactosamine, glucuronic acid and n-acetylneuraminic acid, which do not directly affect agglutination (22), inhibited s e c r e t o r y rosette formation. E r o s e t t e f o r m a t i o n w a s r e d u c e d b y s o m e i n h i b i t o r s , b u t not by o t h e r s , and in s o m e c a s e s the p e r c e n t a g e was a c t u a l l y h i g h e r (Table 4). This suggests that the site of action is not the same for all inhibitors, some acting on cell membrane receptors, some on the s e c r e t o r y a p p a r a t u s , and s o m e on b i n d i n g s i t e s of the a g g l u t i n i n s themselves. C y c l o h e x a m i d e did not affect s e c r e t o r y rosette formation,
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suggesting that agglutinin secretion in itself is not directly affected by inhibition of protein synthesis. More importantly, it suggests that the agglutinins are probably not synthesized during rosette formation but have been pre-formed and stored in the leukocytes, ready to be released after antigen (RBC) stimulation. This premise was implied in the previous observation, using allogeneic rabbit RBC, that restoration of a g g l u t i n i n s y n t h e s i s after d e p l e t i o n did not o c c u r rapidly. Approximately 24 hr were required for reconstitution of the full capacity to secrete a given agglutinin. The evidence suggests a greater degree of specificity than has generally been thought to occur at this phylogenetic level (23,24). Since earthworm hemagglutinins are also known to be bacterial agglutinins (20), such specificity may play a role in r e c o g n i t i o n and r e m o v a l of p a t h o g e n i c o r g a n i s m s and thus be essential to the earthworm's immunodefense system.
REFERENCES i. H a l l , J.L. and R o w l a n d s , D.T., H e t e r o g e n e i t y of l o b s t e r agglutinins. I. Purification and Physicochemical Characterization. Biochem. 13, 821, 1974. 2. Amirante, G.A. and Mazzalai, F.G. Synthesis and localization of hemoagglutinins in hemocytes of the cockroach Leucophaea maderae. Dev. Comp. Immunol. 2, 735, 1978. 3. Boerrigter-Barendsen, L.H., Van den Howven, D.S.P., Sminia, T. and Van der Knapp, W.P.W. Immunocytochemical demonstration of a humoral defense factor in blood cells (amoebocytes) of the pond snail Lymnaea sta~nalis. Cell Tiss. Res. 219, 291, 1981. 4. Vasta, G.R., Cheng, T.C. and Marchalonis, J.J. A lectin on the hemocyte membrane of the oyster (Creassostrea vir@inica). Cell. Immunol. 88, 475, 1984. 5. Cooper, E.L., Evolution of cellular immunity. In: Braun, W. and Ungar, J. (Eds.,) Non-Specific Factors Influencing Host Resistance, Karger, Basel 1973, p. II. 6. Toupin, J. and Lamoureux, G., 1976. Coelomocytes of earthworms: Phytohemagglutinin (PHA) responsiveness. In: Phylogeny of thymus and bone marrow bursa cells (R.K. Wright and E.L. Cooper, Eds.). North Holland, Amsterdam, pp. 19-26. 7. Chateaureynaud-Duprat, P. and Izoard, F. Compared study of i m m u n i t y b e t w e e n two genera of l u m b r i c i e n s : E i s e n i a and Lumbricus. In: Solomon, J.B. and Horton J.D. (Eds.), Developmental Immunobiology, Elsevier/North Holland, Amsterdam, New York 1977, p. 33. 8. Mohrig, W., Kauschke, E. and Ehlers, M. Rosette formation of the Coelomocytes of the earthworm Lumbricus terrestris with sheep erythrocytes. Dev. Comp. Immunol. 8, 471, 1984. 9. Anderson, R.S. Rosette formation by insect macrophages. Inhibition by cytochalasin B. Cell Immunol. 29, 331, 1973. 10. Wright, R.K. and Cooper, E.L. Tunicate leucocyte receptors a n d h e m o l y m p h lectins. In: Cohen E. (Ed.), R e c o g n i t i o n , Proteins, Receptors and Probes: Invertebrates, Alan R. Liss, New York 1984, p. 115.
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Stein, E.A. and Cooper, E.L. Agglutinins as receptor molecules: A phylogenetic approach. In: Cooper, E.L. and Brazier, M.A.B. (Eds.), Developmental Immunology: Clinical Problems and Aging, Academic Press, New York 1982, p. 85. 12. Hartree, E.F. Determination of protein. A modification of the Lowry method that gives a linear photometric response. Aqalyt. Biochem. 48, 422, 1972. 13 T o u p i n , J., Marks, D.H., Cooper, E.L. and L a m o u r e u x , G. Earthworm coelomocytes in vitro. In Vitro 13, 218, 1977. 14 Stein, E. and Cooper, E.L. The role of opsonins in phagocytosis by c o e l o m o c y t e s of the earthworm, L u m b r i c u s terrestris. Dev. Comp. Immunol. 5, 415, 1981. 15 Stein, E.A., Wojdani, A. and Cooper, E.L. Agglutinins in the earthworm, Lumbricus terrestris, naturally occurring and induced. Dev. Comp. Immunol 6, 407, 1982. 16 Young, C.W., Hendler, F.J. and Karnofsky, D.A. Synthesis of p r o t e i n for DNA replication and cleavage events in the sand dollar embryo. Exp. Cell Res. 58, 15, 1969. 17 Schneiderman, M.H., Dewey, W.C. and Highfield, D.P. Inhibition of DNA synthesis in synchronized Chinese hamster cells treated in G1 with cyclohexamide. Exp. Cell Res. 67, 147, 1971. 18 S c h e f l e r , W.C. S t a t i s t i c s for the B i o l o @ i c a l Sciences, Addison-Wesley, Reading 1979, p. 84. 19 Wojdani, A., Stein, E.A. and Cooper, E.L. Agglutinins and proteins in the earthworm, Lumbricus terrestris, before and a f t e r i n j e c t i o n of e r y t h r o c y t e s , c a r b o h y d r a t e s and other materials. Dev. Comp. Immunol. 6, 613, 1982. 20 Stein, E.A.,-Y0unai, S. and Cooper, E.L., In: E.L. Cooper, C. L a n g l e t and J. Bierne (Eds.) 3rd International Congress of Developmental and Comparative Immunolo~y~ Alan R. L1ss, New York, p. 133. 21 Stein, E.A., Avtalion, R.R. and Cooper, E.L. Coelomocytes of the earthworm, Lumbricus terrestris: Morphology and phagocytic properties. J. Morphol. 153, 467, 1977. 22 Stein, E.A. and Cooper, E.L. Carbohydrate and glycoprotein inhibitors of naturally occurring and induced agglutinins in the earthworm Lumbricus terrestris. Comp. Biochem. Physiol. D6B, 197, 1983. 23 Nisonoff, A., Hopper, J.E. and Spring, S.B. The Antibody Molecule, Academic Press, New York 1985, p. 264. 24 Klein, J. ~mmunology. The Science of Self-Nonself Discrimination, John Wiley'and Sons, New York 1982, p. 6. 25 Yeaton, R.W. Lectins of a North American Silkmoth (Hyalophora cecropia): Their Molecular Characterization and Developmental Biology , Ph.D. Th4sis~ ......University of Pennsylvania 1980. Received: June 1987 Accepted: October 1987