Lectin-binding specificities of hemocytes from two strains of Biomphalaria glabrata as determined by microhemadsorption assays

DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. 4, pp. 617-628, 1980

0145-305X/80/040617-12502.00/0 Printed in the USA. Copyright (c) 1980 Pergamon Press Ltd. All rights reserved.

LECTIN-BINDING SPECIFICITIES OF HEMOCYTES FROM TWO STRAINS OF BIOMPHALARIA GLABRATA AS DETERMINED BY MICROHEMADSORPTION ASSAYS 1 1 David A. Schoenberg and Thomas C. Cheng Institute for Pathobiology, Center for Health Sciences Lehigh University Bethlehem, Pennsylvania 18015, U.S.A.

ABSTRACT The effect of eleven lectins on the attachment of human erythrocytes (hrbc) to hemocytes of two strains of Biomphalaria @labrata with differing susceptibilities to Schistosoma mansoni was examined. Con A, RCA-120, and WGA strongly enhanced hemadsorption. RCA-120 and C o n A enhancement were restricted to type A hemocytes. HPA, LCA, TPA, and UEA-60 weakly enhanced hemadsorption. BSA, DBA, PHA-E, and SBA did not enhance hemadsorption. LCA more strongly enhanced hemadsorption with hemocytes from NIH albino strain snails than with hemocytes from 10R-2 strain snails. These findings suggest that gastropod hemocytes with different origins differ quantitatively in their arrays of cell surface oligosaccharides capable of lectin binding.

INTRODUCTION Gastropods have both cellular and humoral means of degrading foreign materials (1-5). Hemocytes, particularly granulocytes, avidly phagocytose small objects as well as form capsules surrounding large objects. The sera of several species of gastropods contain lectins, lytic enzymes, or other factors which enhance phagccytosis (6-9). Strain differences in the structure or function of hemocytes or serum factors may play a role in determining the relative susceptibility of a gastropod strain to a trematode sporocyst.

ipresent address: Marine Biomedical Research Program and Department of Anatomy (Cell Biology), Medical University of South Carolina, P.O. Box 12559 (Fort Johnson), Charleston, South Carolina 29412 U.S.A. 617

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To compare one aspect of hemocyte structure which may be involved in the recognition of foreign objects, we have examined the relative binding capabilities of several lectins to hemocytes from two strains of the planorbid Biomphalaria ~labrata with different susceptibilities to a Puerto Rican strain of Schistosoma mansoni. Lectins display a binding specificity to carbohydrates determined by saccharide structure, sequence and linkage (10-12). We chose to employ a microhemadsorption assay (13) to indicate the ability of the lectins to mediate attachment and endocytosis of live human erythrocytes (hrbcs) to snail hemocytes, and to demonstrate lectin binding to hemocyte surface glycoproteins. MATERIALS AND METHODS Snails: The snails used were our laboratory stocks of i) B. glabrata NIH albino strain (14) (susceptible to our Puerto Ri~an strain of S. mansoni~ and 2) B. glabrata 10R-2 strain (15) (resistant to our strain of S. mansoni). Hrbcs: Typed hrbcs, packed in dextrose-citrate, were stored at ~U C. Immediately prior to use, they were rinsed 3X in Dulbecco's Phosphate Buffered Saline (DPBS), 276 mosmoles (16). Unrinsed hrbcs were discarded after two weeks. Chemicals: Concanavalin A type IV (Con A), erythroagglutinin (PHA-E), lectins from Bandieraea simplicifolia (BSA), Lens culinaris (LCA), Ricinus communis type II-A (RCA-120). Tetra~onolobus purpureas (TPA), l-O-methyl-~-D-galactopyranose (~MG), Nacetyl-D-glucosamine (NAG1), L-fucose, and N-acetyl-D-galactosamine (NAGa) were purchased from Sigma Chemical Co., St. Louis, Missouri. Soybean agglutinin (SBA), wheat germ agglutinin (WGA), and lectins from Dolichos biflorus (DBA) and Ulex europeus (UEA-60) were purchased from Vector Laboratories, Burlingame, California. Lectin from Helix pomatia (HPA) and s-methyl-Dmannopyranoside (eMM) were purchased from Polysciences, Warrington, Pennsylvania. Isolation of hemocytes: Following cardiac puncture, approximately 40 ~i of hemolymph was removed by micropipette from each of 10-15 snails and pooled at 4° C. Fifty ~i of pooled hemolymph was transferred to a microscope slide treated by the method of Niel and Fribourg-Blanc (17) to prevent drying of the preparation. Hemocytes were allowed to attach for 20 min at 24 ° C when the serum was replaced by a hypertonic Chernin's Basic Salt Solution (HS; NaCl, 8.30g/I, other salts, see 18 ), 276 mosmoles, for i0 min. Lectin treatment of hemoc~tes: Attached hemocytes were treated with 50 ~g/ml lectin (HPA: 1 to 400) in HS for 30 min. The cells were then rinsed and maintained in HS for five min before incubation with untreated hrbcs. Lectin treatment of hrbcs: Suspensions of 2 x i0 e hrbcs/ml were treated with 20 ~gTm[ of lectin in DPBS for 30 min with frequent

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LECTIN SPECIFICITY IN MOLLUSCS

v o r t e x mixing. T r e a t m e n t was f o l l o w e d by three w a s h e s b e f o r e i n c u b a t i o n w i t h u n t r e a t e d hemocytes.

619

in DPBS

M i c r o h e m a d s o r p t i o n assay: The HS was d r a i n e d and 250 ~i of a 5 x i0 c e l l s / m l hrbc s u s p e n s l o n was added to the a t t a c h e d hemocytes. A f t e r 30 min, the p r e p a r a t i o n was gently r i n s e d w i t h DPBS. I n c u b a t i o n was c o n t i n u e d in DPBS, w i t h or w i t h o u t added s p e c i f i c l e c t i n inhibitor, for an a d d i t i o n a l 30 min. The prep a r a t i o n was a g a i n g e n t l y rinsed, w i t h two 20 sec t r e a t m e n t s on a r o t a r y s h a k e r at 100 rpm (19), fixed w i t h 2% g l u t a r a l d e hyde in DPBS for 30 min at 4 ° C, r i n s e d and stained w i t h MayG r ~ n w a l d stain. A c o v e r s l i p was m o u n t e d w i t h nail polish. Each w e t m o u n t was e x a m i n e d w i t h b r i g h t field and w i t h N o m a r s k i i n t e r f e r e n c e optics. As u n f i x e d hrbcs w e r e used in this study, h e m o c y t e t r e a t m e n t s and i n c u b a t i o n s w e r e p e r f o r m e d in m e d i a h y p e r t o n i c to snail hemolymph. H e m o c y t e s w e r e t r e a t e d in HS to m a i n t a i n the 0.05% C a C I ~ c o n c e n t r a t i o n n e c e s s a r y for their c o n t i n u e d a d h e s i o n to the -slide. However, hrbc t r e a t m e n t s and i n c u b a t i o n s w e r e perf o r m e d in DPBS, as this s o l u t i o n m i n i m i z e d n o n - s p e c i f i c adh e s i o n of hrbcs to glass. The d e c i s i o n to treat h e m o c y t e s or hrbcs was b a s e d on the s t r e n g t h of h r b c a g g l u t i n a t i o n at a lectin c o n c e n t r a t i o n w h i c h permitted hemadsorption. Hrbcs a g g l u t i n a t e d by Con A and LCA at 20 ~g/ml w e r e r e a d i l y s e p a r a t e d by v o r t e x mixing; hence, h r b c s w e r e t r e a t e d w i t h these lectins. A g g l u t i n a t i o n of hrbcs w i t h H P A at 1:400 d i l u t i o n and all o t h e r lectins at 50 ~g/ml r e s u l t e d in s t r o n g a g g l u t i n a t i o n in w h i c h clumps of hrbcs did not s e p a r a t e as i n d i v i d u a l cells after v o r t e x mixing. Mixing was a c c o m p a n i e d by s u b s t a n t i a l r u p t u r i n g of hrbcs. Hence, hemocytes w e r e t r e a t e d w i t h these lectins. Enumeration: The m i c r o h e m a d s o r p t i o n assay was o r i g i n a l l y int e n d e d to m e a s u r e the a t t a c h m e n t of h r b c s to test cells after l e c t i n t r e a t m e n t (13). As w e w e r e i n t e r e s t e d in a s s e s s i n g the e f f e c t of l e c t i n t r e a t m e n t on the c o m p l e t e p h a g o c y t i c response, i.e., a t t a c h m e n t and e n d o c y t o s i s , we have d i s t i n g u i s h e d b e t w e e n a t t a c h e d and e n d o c y t o s e d hrbcs. E n d o c y t o s e d hrbcs w e r e clearly r e c o g n i z a b l e (Fig. i). The t e r m a s s o c i a t e d is used to d e s c r i b e hrbcs that are either a t t a c h e d to or e n d o c y t o s e d by a hemocyte. F i v e r e p l i c a t e counts of h r b c s a s s o c i a t e d w i t h or e n d o c y t o s e d by 100 h e m o c y t e s w e r e made. S t a t i s t i c a l a n a l y s e s w e r e m a d e u s i n g S t u d e n t ' s t test and the t' test (20). ~ values >0.05 w e r e c o n s i d e r e d not significant. The p e r c e n t a g e s of h e m o c y t e s w i t h a s s o c i a t e d or e n d o c y t o s e d hrbcs w e r e also d e t e r m i n e d .

RESULTS E f f e c t of lectins on a s s o c i a t i o n index: The m e a n number of h r b c s a s s o c i a t e d w i t h 100 h e m o c y t e s from the two strains of B. g l a b r a t a (association index) f o l l o w i n g l e c t i n t r e a t m e n t and i n c u b a t i o n are c o m p a r e d in T a b l e i. In c o n t r o l i n c u b a t i o n s (without l e c t i n treatment), the m e a n a s s o c i a t i o n i n d e x r a n g e d b e t w e e n 2.60 and 7.40. A n average of

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Fig. i. Association of HPA treated B. @labrata NIH albino strain hemocytes with untreated type A hrbcs. May-Gr~nwald stain, green filter, a, hrbc attached to type "A" hemocyte; hrbc endocytosed by type "A" hemocyte.

e,

Fig. 2-3. Control B. glabrata NIH albino strain hemocytes "treated" in lectin-free HS and incubated with type 0 hrbcs. May-Gr~nwald stain. Fig. 2. Group of three fully spread type "A" hemocytes, n, nucleus. Fig. 3. Spread type "B" hemocyte. n, nucleus.

Fig. 4-6. Association of RCA-120 treated B. glabrata NIH albino strain hemocytes with untreated type O hrbcs. Nomarski optics. Fig. 4. Type "A" hemocytes with attached hrbcs. Fig. 5. Low magnification field showing clumped distribution of hrbcs restricted to hemocytes. Fig. 6. Adjoining type "A" and type "B" hemocyte, showing differential binding of hrbcs to the two cell types. A second type "B" hemocyte is also shown, a, type "A" hemocyte; b, type "B" hemocyte.

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Table 1 E f f e c t of lectins on h e m o c y t e - h r b c a s s o c i a t i o n index a Treatment

Blood type

Lectin

A s s o c i a t i o n index (hrbc/100 hemocytes) NIH a l b i n o

hrbc

0+

None

(5.80+

Con A

hemocyte

O+

(2.60+

1.41)

584.60+ 28.21"**

LCA

52.80+ 32.51 **c

17.60+ ii.73 **c

None

(4.40+

3.21)

(6.80+

3.42)

6.00+

4.12

1.00+

1.22"

RCA-120

567.10+127.78"**

508.67+

8.08***

TPA

29.00+

5.34***

21.20+

UEA-60

25.40+

5.41"**

26.20+ Ii.01"*

WGA A+

3.96)

561.40+ 50.30***

PHA-E

hemocyte

10R-2 b

271.80+ 30.75***

8.53**

298.20+ 33.36***

None

(5.60+

2.07)

(7.40+

1.34)

BSA

10.40+

4.88

4.20+

4.97

DBA

3.60+

2.61

2.00+

0.71"

HPA

37.40+

SBA

6.20+

4.98***

28.00+ 10.25

2.86

4.20+

2.77*

m

ahrbc c o n c e n t r a t i o n :

5 x 10 ~ c e l l s / m l

b M e a n ~ S.D. Cstrain difference: (t' test)

*~

<

**~ < 0.05

< ~

< 0.01

***~ <

.05 .01 .001

3% of a s s o c i a t i n g hrbcs w e r e e n d o c y t o s e d . Two m o r p h o l o g i c a l l y d i s t i n g u i s h a b l e types of h e m o c y t e s w e r e recognized. W h e n fully spread, h e m o c y t e s w i t h m o r p h o l o g y A bore n u m e r o u s f i l o p o d i a (Fig. 2). The n u c l e u s was flattened, m o r e d a r k l y s t a i n e d than the cytoplasm, and o f t e n c o n t a i n e d v i s i b l e chromatin. Hemocytes w i t h m o r p h o l o g y B w e r e r o u n d e d and had few, short filop o d i a (Fig. 2, 3). T h e r e was o f t e n a r a i s e d area of c y t o p l a s m above the nucleus. BSA, DBA, PHA-E, and SBA t r e a t m e n t did not s i g n i f i c a n t l y enh a n c e the a s s o c i a t i o n index above the c o n t r o l value for the p a r t i c u l a r b l o o d type and snail s t r a i n (Table I). Indeed, the a s s o c i a t i o n index was s i g n i f i c a n t l y lower than that of the controls a f t e r DBA, PHA-E, and SBA t r e a t m e n t of 10R-2 h e m o c y t e s (0.05 < ~ < 0.01). A s s o c i a t i o n indices r a n g e d from 1.0 to 10.4. HPA, LCA, TPA, and UEA-60 t r e a t m e n t s s i g n i f i c a n t l y e n h a n c e d the a s s o c i a t i o n i n d e x c o m p a r e d to controls, w i t h m e a n indices r a n g i n g b e t w e e n 17.6 - 52.8 (~ < 0.01). The p e r c e n t a g e s of ass o c i a t i o n w i t h these t r e a t m e n t s r a n g e d from 13.5 to 33.5%.

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Con A, RCA-120, and W G A s t r o n g l y e n h a n c e d the a s s o c i a t i o n index. M e a n a s s o c i a t i o n indices r a n g e d from 271-616. More than 90% of the h e m o c y t e s w e r e a s s o c i a t e d w i t h hrbcs. Distribution of hrbcs among h e m o c y t e s , however, was not uniform, e.~., after Con A t r e a t m e n t of hrbcs, 5% of N I H a l b i n o h e m o c y t e s b o u n d no hrbcs, 32.2% b o u n d 1-4 hrbcs, 45.4% b o u n d 5-8 hrbcs, 15.2% b o u n d 9-12 hrbcs and 2.2% b o u n d > 13 hrbcs. Type A h e m o c y t e s treated w i t h RCA-120 or i n c u b a t e d w i t h h r b c s t r e a t e d w i t h Con A r e m a i n e d f l a t t e n e d b e n e a t h a t t a c h e d hrbcs (Fig. 4). C l u m p s of hrbcs w e r e found a t t a c h e d to h e m o c y t e s but not a t t a c h e d d i r e c t l y to glass (Fig. 5). A t t a c h m e n t of hrbcs to RCA-120 t r e a t e d type B h e m o c y t e s was m u c h less than a t t a c h m e n t to type A h e m o c y t e s (Fig. 6). C l u m p i n g of hrbcs was not o b s e r v e d w i t h type B cells, even if a d j a c e n t to a type A cell w i t h a t t a c h e d hrbcs. This i n d i c a t e s a d i f f e r e n t i a l r e s p o n s e of these two c a t e g o r i e s of cells to RCA-120. Similarly, clumps of Con A treated hrbcs w e r e r e s t r i c t e d to type A h e m o c y t e s . Both A and B h e m o c y t e s b o u n d W G A - t r e a t e d hrbcs. The only s i g n i f i c a n t d i f f e r e n c e in a s s o c i a t i o n index b e t w e e n strains r e s u l t e d from LCA t r e a t m e n t of hrbcs (Table i). NIH albino strain h e m o c y t e s b o u n d 3X as m a n y t r e a t e d hrbcs as did 10R-2 strain h e m o c y t e s . This suggests that the n u m b e r of b i n d i n g sites of c e r t a i n lectins on h e m o c y t e s may d i f f e r b e t w e e n strains of B. glabrata. S a c c h a r i d e i n h i b i t i o n of lectin effect: A d d i t i o n of s a c c h a r i d e w h i c h s p e c i f i c a l l y b i n d s - t o the l e c t i n d e c r e a s e d the a s s o c i a t i o n index (Table 2). A d d i t i o n of s p e c i f i c i n h i b i t o r s to i n c u b a t i o n s w i t h HPA, LCA, TPA, and UEA-60 t r e a t e d cells l o w e r e d the ass o c i a t i o n index to control values. A d d i t i o n of s p e c i f i c inh i b i t o r s to i n c u b a t i o n s with Con A, RCA-120, and W G A t r e a t e d cells d e c r e a s e d the a s s o c i a t i o n index to 9%, 18%, 32%, r e s p e c t ively, of values o b t a i n e d w i t h o u t addition. These, however, r e m a i n e d above control values (~ < 0.001). These observations indicate that l e c t i n - e n h a n c e d h e m a d s o r p t i o n results from s p e c i f i c lectin b i n d i n g to s a c c h a r i d e s on the surface of b o t h h e m o c y t e and hrbcs. Endocytosis: The n u m b e r of e n d o c y t o s e d hrbcs in h e m o c y t e prep a r a t i o n s was small (Table 3). Less than 5 hrbcs w e r e e n d o c y tosed per i00 h e m o c y t e s in all p r e p a r a t i o n s l i s t e d in T a b l e s 1 and 2, w i t h or w i t h o u t added l e c t i n inhibitor. In some cases, this l i m i t e d e n d o c y t o s i s may r e f l e c t the short p e r i o d (30 min) b e f o r e the first rinse. W h e n RCA-120 t r e a t e d h e m o c y t e s w e r e i n c u b a t e d w i t h a 5 x 10 ~ h r b c s / m l s u s p e n s i o n (i/i0 c o n c e n t r a t i o n of o t h e r experiments) for 60 min b e f o r e r i n s i n g and fixing, a m e a n of ii of 61 a s s o c i a t e d hrbcs (18%) w e r e e n d o c y t o s e d by i00 hemocytes. W i t h the 30 m i n rinse included, a m e a n of 0.8 of 20 a s s o c i a t e d hrbcs (4%) w e r e e n d o c y t o s e d by i00 h e m o c y t e s . These p e r c e n t a g e s are s i g n i f i c a n t l y different, (arc sine t r a n s f o r m , t s statistic, (20). However, for control, Con A, or W G A treatments, neither this p e r c e n t a g e nor a s s o c i a t i o n index v a r i e d w i t h treatment. This suggests that the time course of l e c t i n - m e d i a t e d a t t a c h m e n t and e n d o c y t o s i s varies w i t h the l e c t i n used.

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Table

Vol. 4, No. 4

3

Endocytosis by NIH albino strain hemocytes after RCA-120 and WGA treatment Endocytosis

T r e a t m e n t a'b Control,

30 min e

0.40 + 0.55

Control,

60 min

0.60 + 0.89

Con A,

30 min

1.60 + 2.30

Con A, 60 min

1.20 + 1.30

RCA-120,

2.22 + 1.99

WGA,

30 min

30 min

ahemocyte

4.00 + 2.34

treatment,

bhrbc concentration: CEndocytosis

index c'd

index:

type 0 + hrbc 5 x l0 T cells/ml hrbcs e n d o c y t o s e d / 1 0 0

hemocytes

dMean + S.D. eIncubation

time

DISCUSSION The results of this study demonstrate that several w e l l - c h a r acterized lectins enhance a t t a c h m e n t of hrbcs to spread B. @labrata hemocytes during s h o r t - t e r m m i c r o h e m a d s o r p t i o n assays. A t t a c h m e n t is partially or completely reversible upon a d d i t i o n of a small saccharide w i t h high b i n d i n g affinity for the lectin. L e c t i n m e d i a t e d attachment thus results from specific lectin binding to carbohydrate moieties on the surface of both cells. Incomplete r e v e r s i b i l i t y of e n h a n c e d attachment may result from the steric u n a v a i l a b i l i t y of some l e c t i n - g l y c o p r o t e i n bonds or from the d e v e l o p m e n t of n o n - l e c t i n requiring i n t e r c e l l u l a r interactions before the saccharide was added (21). Several properties of the lectins and of the cell surfaces may contribute to the differential ability of these lectins to mediate h e m a d s o r p t i o n (22). The number of subunits of the lectin determines its ability to induce m a c r o p h a g e v a c u o l a t i o n and to cross-link its b i n d i n g sites w i t h i n the plane of the cell membrane (23)- C a r b o h y d r a t e binding sites of lectins with the same specificity to simple saccharides may vary in b i n d i n g perm i s s i v e n e s s to extended lectin binding sites on g l y c o p r o t e i n s (24). The p o s i t i o n along the o l i g o s a c c h a r i d e side chain of membrane binding sites may determine the extent of their lectininduced cross-linkage (22). A difference in the number and d i s t r i b u t i o n of lectin b i n d i n g sites on either i n t e r a c t i n g cell may result in a striking difference in the endocytosis index (23). Yeast, with a cell wall rich in e - D - m a n n o p y r a n o s y l residues with 1+2 and 1+3 linkages (25), will bind, at saturation, 3X as many Con A molecules as do erythrocytes (23, 26). After identical treatment (60 min uninterrupted incubation), a mean of 1.2 Con A treated hrbcs were

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endocytosed by 100 NIH albino strain hemocytes (Table 3), whereas a mean of 108 Con A treated yeast cells were endocytosed by i00 hemocytes from the same strain (27). The greater density of Con A binding sites on yeast can provide the greater number of intercellular lectin-mediated linkages, which may be required if endocytosis is to follow attachment (23). This study was undertaken to compare the response of hemocytes from planorbid strains differing in schistosome susceptibility to lectin-mediated hemadsorption. Hemocytes from these two B. ~labrata strains differed only in response to LCA treated hrbcs. There was no difference in response to hrbcs treated with Con A, another lectin displaying specificity for D-mannose and D-glucose. These two lectins differ in number of subunits and in binding constants for simple saccharides (28). LCA binds to a more restricted set of glycoproteins than Con A (24). Hence, LCA may be more sensitive to subtle differences in this class of extended lectin binding sites of hemocyte glycoproteins. Another difference in lectin response was observed between two morphologically distinguishable categories of B. glabrata hemocytes. RCA-120 and Con A strongly enhanced hrbc attachment to hemocytes bearing long filopodia and which may gradually retract from the substrate. Hrbc attachment to hemocytes bearing short filopodia and which remained spread was not enhanced. These morphologically distinct cells thus differ in surface structure. In a similar way, behavior after lectin treatment can distinguish subpopulations of oyster hemocytes (29,30). A similar pattern of lectin binding has been observed with hemocytes from the helicid, Helix pomatia. These hemocytes agglutinate with and form hrbc rosettes with lectins which strongly enhance hemadsorption by B. ~labrata hemocytes, but not with weakly enhancing or nonenhancing lectins (31-32). Species, strain, and individual differences have also been observed in the structure or activity of gastropod lectins and blood group substances (33-36). It is possible that such differences in lectin and hemocyte glycoprotein structure may be complementary. ACKNOWLEDGEMENTS The first author is an NIH Postdoctoral Research Fellow (AI-I05393-02). This work was also supported in part by NSF grant PCM-7723785. We thank the Miller Memorial Blood Center, Bethlehem, Pennsylvania, for providing the human erythrocytes used in this investigation. REFERENCES i.

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