Monoclonal antibody recognized hemocyte subpopulations in juvenile and adult Lymnaea stagnalis: Functional characteristics and lectin binding

DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. 12, pp. 17-32, 1988 0145-305X88 + .00 Printed in the USA. Copyright (c) 1988 Pergamon Journals Ltd. All rights reserved

MONOCLONAL A N T I B O D Y RECOGNIZED HEMOCYTE S U B P O P U L A T I O N S J U V E N I L E AND ADULT LYMNAEA S T A G N A L I S : F U N C T I O N A L C H A R A C T E R I S T I C S AND L E C T I N B I N D I N G

Ronald

IN

Dikkeboom, Jolanda M.G.H. Tijnagel and Wil P.W. van der Knaap

Laboratory of Medical Parasitology, Free University, P.O. Box 7161, The Netherlands

Faculty of Medicine, 1 0 0 7 MC A m s t e r d a m ,

ABSTRACT The mouse monocIonal antibody LS1 r e c o g n i z e s a membrane epltope present on circulating hemocytes of the gastropod mollusc Lymnaea stagnalis. In both Juvenile and adult pond snails, LSI+ (LS1 positive) hemocytes have the morphology of immature cells. The percentage of LSI+ hemocytes is higher in juveniles (ca. 39%) t h a n it is in adults (ca. 14%). Functional characteristics of LSl+ hemocytes and lectin binding to these cells were studied. In both age groups, the proliferative activity, as measured by the incorporation of deoxybromouridine, is much higher for LSI+ hemocytes than it is for LSI(LSl negative) ceils. LSI+ hemocytes are phagocytioal ly less active and have a lower lysosomal enzyme (peroxidase) content as compared to hemooytes that lack the epitope. Histochemical staining of the total population of circulating hemocytes shows that the lectlns DBA, B S - I - A 4 and BS-I-B4, PNA, SBA and ECA do not react with the hemocytes. L T A , APA, WGA, Con A and LCA bind to all hemocytes. RCA and STA recognize surface carbohydrate moieties present on subpopulatlons of hemooytes only. The LSI+ hemocyte population virtual ly lacks the carbohydrate residues recognized by STA, whereas the LS1- population never ShoWS b i n d i n g o f RCA. Our results support the findings that t h e LS1 e p i t o p e is a membrane marker of less differentiated hemocytes in both juvenile and adult L.stagnalis. Furthermore, they suggest a correlation between the presence of t h e LS1 epitope and the absence o f STA b i n d i n g , whereas absence o f t h e LS1 m a r k e r may c o r r e l a t e with the presence of a sugar recognized b y RCA.

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INTRODUCTION Gastropod molluscs possess an effective internal detente system that is Capable of deal ing with a variety of invading organisms (see Sminia 1981; Bayne 1983). The system comprises humoral factors and several types of defence cel Is: antigen-trapping cel Is, fixed phagocytes, resident or tissue hemocytes and circulating hemocytes. In freshwater snails, the circulating hemocytes are important defence cel Is; they are involved in wound heal ing, phagocytosis, encapsulation and the production of several types of humoral factors (see Sminia and Van der Knaap 1986). The population of circulating hemocytes is heterogeneous: cells of e.g. Biomphalaria glabrata a n d Lymnaea s t a g n a l l s vary in morphology, enzyme contents, level of defence activity and lectin binding (Cheng 1975; Renwrantz and Cheng 1977; Yoshino 1983; Dikkeboom et al. 1984; 1985b). Moreover, by applying monoclonal antibodies as membrane probes it was assessed that circulating hemocytes of B.glabrata constitute an a n t l g e n i c a l l y heterogeneous population (Yoshino and Granath 1983). The same authors showed that hemocytes bearing the so-called BGH1 e p i t o p e are reduced in both their phagocytic and lysosomal acid phosphatase activities as compared to cells lacking t h e BGH1 s u r f a c e marker (Yoshino and Granath 1985). In a p r e v i o u s p a p e r we r e p o r t e d the production of nine monoclonal antibodies (LS1 - L S 9 ) d i r e c t e d against hemocytes of the pond snail L.stagnalis and described the morphology of several antigenical ly distinct subpopulations (Dikkeboom e£ a l . 1985a). In the present study, we f o c u s e d o n t h e moAb LS1 b e c a u s e of two interesting properties: the epitope recognized by this monoclonal is localized o n t h e h e m o c y t e m e m b r a n e , a n d LS1 r e a c t s with a subpopulation of hemocytes of which the proportion differs for juvenile and adult snails. Some f u n c t i o n a l characteristics (proliferative activity, phagocytosis and lysosomal enzyme content) of the LSI+ hemocytes and LSlcells are compared; also the presence of surface carbohydrate residues on the hemocytes Is studied by applying lectlns as specific probes. Differences between hemocyte subpopulations In j u v e n l l e and adult L.stagnalls are discussed and age-related variations evaluated.

MATERIALS

AND METHODS

Snails Juvenile (shell height 10 ± 1 mm) a n d a d u l t (shell height 25 ± 1 mm) s p e c i m e n s of the pond snail Lymnaea s t a g n a l l s were obtained from the Biological Laboratory of the Free University, Amsterdam. The animals were kept and fed as described previously (Dlkkeboom eta/. 1984). Hemolymph collection and hemocyte monolayers The Col lection of hemolymph and the preparation of hemocyte monolayers were essential ly as described by Smlnia e t a l . (1979). Hemolymph was extruded from juveni le and adult L.stagnalls and hemocyte monolayers were made on CL-IO0 microprint stock slides (Nutacon, The Netherlands) by incubation for 30 m i n a t room temperature (RT) in a moist atmosphere.

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Immunostainin~ with monoclonal antibody LS1 Hemocyte monolayers were rinsed carefully with snail Sal Ine (of. Sminla et al. 1979). The hemocytes were fixed for 1 h at RT w i t h a mixture of 1% p a r a f o r m a l d e h y d e and 0.5% glutaraldehyde in a 0.03 M Na-cacodylate buffer (pH 7 . 4 ) . The slides were washed in 0.05 M Tris-buffered 0 . 9 % NaCl s o l u t i o n ( pH 7 . 4 ) ; this buffer was used in all wash and dilution steps, unless indicated otherwise. Free aldehyde groups were blocked by incubation in Tris-HCI containing 0.02 M glycine. For immunohistochemistry, a routine indirect immunoperoxidase staining was performed as follows. The hemocytes were incubated for 30 m i n with the undiluted culture supernatant containing mouse-anti-hemocyte monoolonal antibody LS1 ( D i k k e b o o m et al. 1985a). In the second step peroxidaseconjugated goat-anti-mouse Ig ( N o r d i c , The Netherlands; diluted 1:50, incubation 30 m i n ) was u s e d , and a m i x t u r e of 3,3' diaminobenzidine tetrahydrochloride ( D AB, 0 . 5 m g / m l ; BDH C h e m i c a l s Ltd, UK) a n d H 2 0 2 ( M e r c k , FRG; 0 . 0 1 % ) served as substrate (10 min). Slides were washed, dehydrated in a series of ethanol and mounted i n DePeX ( B D H ) . E n d o g e n o u s peroxidase activity (EPO) p r e s e n t in the hemocytes was el iminated by pre-incubation i n 100% m e t h a n o l with 0.02% H202 for 10 m l n . For immunofluorescence, tetramethylrhodamine isothiocyanate (TRITC) conjugated rabbit-anti-mouse Ig (DAKQ, D e n m a r k ; diluted 1:50) was u s e d in the second step (30 min, RT). Slides were coversl ipped with Tris/glycerol (1:1) and the edges sealed with nail polish. In control slides, moAb LS1 was replaced by Tris-HCl o r an i r r e l e v a n t culture supernatant. The morphology of LSl+ and LSlhemocytes was studied light microscopical ly; immunofluoresoence slides were examined using a Zeiss fluorescence (photo)microscope equipped with rhodamine filter sets and phase contrast optics. The percentage of LSl+ cells was determined by observing, at random, 1000 h e m o c y t e s on five different slides. Proliferative activity The proliferative activity of LSI+ and LS1- hemocytes was studied by using an immunohistochemical double staining technique (of. Harms et al., in press). Proliferative acttvity in vitro was demonstrated by Incorporation of bromodeoxyuridlne (BrdUrd) by the hemocytes; BrdUrd, a thymidlne analogue, is incorporated Into reduplicating DNA. F o r s u b s e q u e n t immunoenzymatlc staining with a monoclonal antibody detecting BrdUrd, DNA h a s t o be d e n a t u r a t e d by means of acid hydrolysis. For BrdUrd Incorporation, hemolymph from. juvenile or adult snails was pooled in snail sal Ine to which 5-bromo-2'-deoxyuridine (Sigma, USA; f i n a l concentration 10 #M) was added. Monolayers were made and cel Is were left f o r 30 m i n in the BrdUrd containing sal ine in a moist atmosphere a t RT. T h e n , the hemocytes were rinsed and fixed with a mixture of glutaraldehyde-paraformaldehyde as described above. Endogenous peroxidase was inactivated by incubation in 100% m e t h a n o l with 0.02% H202 for 10 m i n . The slides were rinsed in Tris-HCI a n d an immunoperoxidase staining with moAb LS1 w a s p e r f o r m e d essentially as described above. LSI+ cells were visual ized by developing the peroxidaseconjugate in a 0.1M Na-acetate buffer (pH 5.0) which contained 3-amino-9-ethylcarbazole (0.2 mg/ml ; Sigma) and 0.01% H202 (of. Graham et al: 1965). Incorporated BrdUrd was subsequently demonstrated as follows: after washing in distilled water, the monolayers were rinsed i n 1 N HCI a t RT f o r 1 mln,

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hydrolyzed in 1N HCl a t 6 0 °C f o r 15 m i n , rinsed again In 1N HCI a t RT f o r 1 m l n , and w a s h e d In T r i s - H C l . Then, the cells were incubated for 30 min with 1:10 diluted antl-bromodeoxyuridlne monoclonal antibodies (anti-BrdUrd; Euro-Dlagnostlcs, The Netherlands). After washing in Tris-HCl, slides were Incubated with peroxldase-conjugated goat-anti-mouse Ig. The reaction product was visualized using a DAB / H 2 0 2 m e t h o d w i t h cobalt and nickel Ions (cf. De J o n g e t a/. 1985). SI i d e s w e r e c o v e r s l l p p e d with Aquamount (BDH) and the correlation of both labels was studied. In control slides, hemocytes were either stained Immunohistochemical ly with moAb LS1 or they were Incubated with BrdUrd and stained with anti-BrdUrd as described above. Endogenous peroxldase (EPO) In order to investigate possible correlations between the presence o f t h e LS1 e p i t o p e on the hemocytes and their endogenous peroxldase contents, a combination of Immunohlstochemlcal and enzyme-hlstochemical techniques was used. First, to local Ize the enzyme peroxidase, hemocyte monolayers were incubated with 0.05 M Trls-HCI (pH T.6) containing 4-chloro-l-naphthol (0.6 mg/ml; Sigma) and H202 (0.01%) for 10 m i n a t RT ( c f . Nakane 1968). The presence of EPO w a s thus revealed by a blue reaction product. T h e n , on t h e same monolayers an immunoperoxldase staining was performed with t h e moAb L S 1 . To g e t a c l e a r color contrast with the enzyme staining the peroxidase-conjugate was developed in a Na-acetate buffer containing 3-amino-9-ethylcarbazole a n d H202 as described above (c{. G r a h a m e£ a l . 1965); the presence o f t h e LS1 epltope on snail hemocytes was thus visual ized by a red color. Sl i d e s were mounted in Aquamount and studied light microscopically. Of about 1000 hemocytes from both juvenile and adult snails the number of LSI+ and/or EPO+ c e l l s was determined. Phagocytosls To s t u d y a possible correlation between the binding o f t h e moAb LS1 to L.stagnalls hemocytes and their phagocytic capacity, Immunohlstochemlstry was combined with in vitro phagocytosis assays. For phagocytosis, (non-fixed) monolayers of hemocytes of juvenile and adult snal Is were Incubated in vitro with formalinfixed rabbit red blood cells (RRBC; 2% v / v In snal I saline) a t RT for 1 h (cf. Dlkkeboom et al. 1985b). E x c e s s RRBC w e r e w a s h e d o f f with snal I saline. Hemocytes were fixed and an i n d i r e c t immunoperoxidase staining with moAb LS1 was performed as described above. SI I d e s w e r e d e h y d r a t e d , mounted i n DePeX a n d s t u d i e d light microscopically. Approximately 1000 hemocytes (five slides) were examined and the percentages of LSl+ and phagocytical ly active cells were determined. Lectin histochemistry In order to study differences between L.stagnalis hemocytes associated with surface carbohydrates, we u s e d 13 l e c t i n s as specific probes. The lectins used in this study are listed in table 1, t o g e t h e r with their acronyms, major sugar speciflcltles, and corresponding inhibition sugars used. Lectins were either conjugated with peroxidase or with fluorescein isothlocyanate (FITC); all lectins and sugars were obtained from Sigma.

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Table used in

Lectins

Lectin common acronym orig{n name .......................................................................

1 this

21

study

major Sugar specificity

Inhibition sugar

Abrus precaCorius

jequirity bean

APA

D-Gal

D-Gal

Arachis hypogaea

peanut

PNA

GaI-~-(1-3)-GaINAc

o-methylgalactoside

Bandeiraea simplictfolia

griffonia

BS

I-A 4

O-D-GarNAC

o-O-GalNac

BS

I-B 4

G-D-Gal

Q-D-G]cNac

a-D-GIc,o-D-Man

Q-D-GIc

Con

jack

Dotichos biflorus

horse

gram

OBA

O-D-GalNAc

O-D-GalNac

EryChclna cristagalli

coraE

tree

ECA

D-Gal

lactose

soybean

SBA

Q-D-GalNAc,~-D-Gal

a-D-GalNac

common lentil

LCA

Q-D-GLc,~-D-Man

mannose

RCA

~-D-Gal

STA

(D-GLcNAc)

pea

LTA

O-L-FUC

germ

WGA

(~(1-4)-D-GIcNAc)

Gfycine Lens

max

culinaris

RicJnus communis

castor

Solarium tuberosum

potato

Tetragonolobus purpureas

winged

TriCIcum vulgarls

wheat

Lectin FUC

concentrations:

bean

A

Canavalia ensiformis

bean

0.1

mg/mt

= ~uoose, Gal = galactose, GIcNAc = N-acetyl-glucosamine,

Inhibition

lactose

chitin

2

Q-L-FUC

Sugar

2

D-GIcNac

concentrations:

0.1M

GalNAc = N-acetyl-galactosamine, GIc = glucose, Man = mannose

Hemocyte monolayers of juveni e and adult L.stagnalis were fixed and incubated with the ectins (final concentration: O.1 mg/ml Tris-HCI) for 30 min at RT. After washing with Tris-HCl, slides that had been treated w th peroxidase-conjugated lectins were developed with DAB/H202, dehydrated and mounted in DePeX; slides treated with FITC-con ugated lectins were mounted in Tris/glycerol . In controls, lectins were incubated with their corresponding inhibiting sugars (final concentration: 0.1 M in Tr is-HCI) for 10 m i n a t RT before application to the hemocyte monolayers. The leotins STA a n d RCA r e a c t e d with only a part of the hemocyte population. Therefore, STA and RCA were subsequently used in double (immuno)staining techniques to search for a possible correlation between surface carbohydrate composition and the presence of t h e LS1 epitoPe on the hemocytes. For this, fixed hemocyte monolayers were first incubated with a FITC-conjugated lectin and subsequently stained with moAb LS1 and TRITCconjugated rabbit-anti-mouse Ig (for methods, see above). Reversed stain;ng sequences were applied to check for mutual hindrance and/or competition by the probes used. Slides were processed as described above and studied with a Zeiss fluorescence (photo)microscope equipped with FITC and rhodamine filter sets, and phase contrast optics.

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1+ L

N

Figure 1 Hemocytes from Juvenile L. stagnalis after immunoperoxidase staining with moAb L S 1 . Note the difference in morphology of LSI+ (dark y stained) and LSI(unstained) hemocytes. N nucleus; L Iobopod; X 1100.

RESULTS

LS1-immunosta

nin~

immunof luorescent staining corroborate our ImmunoPeroxldase and a/. 1985a) that the moAb LS1 earlier findings (Dikkeboom et s present on the membrane of recognizes an antigen that hemocytes are stained; with moAb L.stagnalls hemocytes. Not all detect antigenlcal ly distinct LS1 it is possible to le and adult snails. Marked subpopulations in both juven number of LSI+ hemocytes of both differences exist between the age groups; of the cells from Juvenile snails 39.3% ± 10.4 (mean ± SD) reacted with the monoclonal antibody, whereas only 14.0% ± 6,4 of the hemocytes from adults expressed t h e LS1 e p i t o p e . The morphology of the positive cells was not much different in the two age groups. In general, the LSI+ hemocytes were relatively smal I cel Is with a short Iobopod (figure 1); occasional iy, LSI+ hemocytes with branched pseudopodia were observed in hemolymph from adults. The LSIpopulation of juvenile snails Is heterogeneous: both round and spreading cells were found; in adults, LSI- hemocytes were virtual ly all large, spreading cells. Proliferative activity The results of the In vitro SUbSequent double Immunostalnlng a r e shown In f i g u r e 2.

incorporation of BrdUrd and the with moAb LS1 and antl-BrdUrd

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100

90

8 o cL

BrdU]uven~Je

80 70

adult

8rdU-

60 50 4-0 30 20 10 0 LS1 +

LS1 -

LS1 +

LS1 -

Figure 2 The presence o f t h e LS1 e p i t o p e on hemocytes from juvenile and adult L.stagnalis in relation to the proliferative activity of the cells as measured by the incorporation of bromodeoxyuridlne (BrdU).

The maJority incorporated respectively, In both age activity.

of the LSI+ hemocytes of juvenile adult snails had BrdUrd ( 2 3 % a n d 7% o f t h e t o t a l hemocyte Population, in contrast to 9% a n d 5% L S l + / B r d U r d hemocytes). groups, LSIhemocytes hardly showed proliferative

Peroxidase contents The relation between the expression of t h e LS1 e p i t o p e and the activity of endogenous peroxidase of the hemocytes of both age groups is shown in figure 3. In juvenile snails, c a 31% o f t h e total population was LSI+; of these 3% s h o w e d EPO a c t i v i t y and 28% w a s EPO-. Of the LS1- hemocytes from juveniles (89%), a much larger proportion was EPO+ (46%). In adults, ca 13% of the hemocytes was LSI+; 3% w a s L S I + / E P O + a n d 10% L S I + / E P O (see also figure 4). Of the LSI- cells, virtually all (83% out o f 87%) contained the enzyme peroxidase. Pha~ocytosls The correlation between the binding of the moAb LS1 to L.stagnalls hemocytes and their phagocytic capacity is shown in figure 5. Ca 29% of the hemocytes from juveni le snai Is reacted with moAb LS1 I n these assays; of these, 4% w a s LSI+ and had phagocytosed RRBC, whereas 25% w a s L S I + and non-phagocytic. In contrast, more LSIhemocytes were phagocytical ly active (29%); the remaining 42% o f t h e t o t a l population lacked t h e LS1 e p l t o p e and was non-phagocytic. In adUlts the percentages of phagocytic and non-phagocytic hemocytes which b o u n d t h e moAb LS1 w e r e 3% a n d 7%, r e s p e c t i v e l y ; of the LSIpopulation a higher proportion was phagocytical ly active (46% out of 90%).

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l

100 9O El=O+ 80

o

&

adult

juvenile

70

o

60 50

EPO+

u 40 -5 30

EPOEI=O -

EPOEPO-

VPO+ 0

/ / / LS1 +

to

LS1 -

LS1 +

LS1 -

Figure 3 The expression Of t h e LS1 e p i t o p e on hemocytes from juveni le and adult L.stagnalis in relation endogenous peroxidase (EPO) a c t i v i t y of the cells.

Lectin histochemistry The lectins B S - I - A 4 , B S - I - B 4 , DBA, ECA, PNA a n d SBA d i d n o t s h o w any binding to hemocytes of juvenile or adult L.stagnalis. Using APA, Co n A, LCA, LTA o r WGA a l l hemocytes of both age groups were stained. In all cases binding was specific as seen from inhibition with corresponding carbohydrates. APA a n d LTA s h o w e d moderate staining; staining by these lectins demonstrates the presence of, respectively, galactosyl and fucosyl residues on t h e hemocyte surface. Con A a n d LCA S h o w e d s t r o n g binding, indicating the existence of mannosyl and glucosyl groups on the hemocyte membrane. The binding of WGA t o L.stagnalls hemocytes Indicates that N-acety gluoosaminyl residues are present on the hemocyte glycocalyx. The lectins RCA ( m a j o r sugar specificity: D-galactose) a n d STA (major spec ficity: N-acetyl glucosamine ol Igomers) did stain hemocytes, but not al I. The percentage of hemocytes binding RCA was about twice as high in juvenile snal Is ( 2 4 % ) a s I t w a s tn adults (10%); after staining with STA n o m a r k e d differences were observed between the percentage of positive cells from both age groups (94% a n d 96%, r e s p e c t i v e l y ) . The results of the combined staining with each of the lectlns RCA or STA and moAb LS1 are shown in figures 6 and 7. A clear distinction could be made between hemocytes reacting with FITCconjugated lectin (green fluorescence), LSI+ cells (red fluorescent TRITC-conjugate), hemocytes that were stained with both probes (green and red; see figure 8) and cells that were negative for both labels (cells visible with phase contrast).

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LSI+

25

"

Figure 4 Hemocytes from adult L.stagnalis stained for endogenous peroxidase (EPO) a c t i v i t y and t h e LS1 e p i t o p e . N o t e t h e a b s e n c e o f EPO i n the LSI+ hemocyte (darkly stained) and the presence o f EPO+ g r a n u l e s (arrows) in LSI- cel Is. N nucleus; X 1100.

Concerning the binding o f RCA, t h e LSI+ populations of both age groups were heterogeneous: RCA+ c e l I s ( 2 4 % i n J u v e n i l e s a n d 10% in adults) a n d RCA- h e m o o y t e s (4% I n J u v e n i l e s a n d 2% In adults) were observed; the LSIpopulations, however, were consistently negative for this leotln. W i t h STA t h e o p p o s i t e seems to be the case: in the juvenile and adult LSIpopulation STA+ ( 6 3 % a n d 87.5%) and STA- hemocytes (6% a n d 3 . 5 % ) a r e p r e s e n t ; in juveniles LSI+ hemocytes did not react with STA, whereas in adults the percentage of LSI+/STA+ cells was low (0.5%). Reversing the order of appl ication of the lectlns and the monoclonal antibodies (i.e. moAb LS1 b e f o r e the lectlns) did not affect the staining results.

DISCUSSION The present study concerns the heterogeneity of circulating hemocytes from juvenile and adult specimens of the pond snail L,stagnalis. Using both monoclonal antibodies and lectins as probes, It Is possible to distinguish several hemocyte subpopulations that bind one, both or none of the probes. Some o f these subpopulations differ from each other with respect to morphological and functional characteristics. Immunostaining shows that t h e moAb LS1 d o e s n o t b i n d to the cell membrane of al I hemocytes of Juveni le and adult L.stagnalls, thus corroborating results of our previous study (Dlkkeboom eta/. 1985a). This suggests that moAb LS1 recognizes a hemocyte subpopulation differing in antigenic surface determinants from LSIhemocytes.

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100 90

8

80

&

70

o

juvenile

adult

6O 5O

phag+ h 777..~..p7 p o g -

phag -

,.30

NN

phag+

~ / / / / / , \ \ \ \ " ,~

,/////, , \ \ \ \ ~ , ,/////~ ,/////,

10

phog

pho~+ ~ LS1 +

I

-

,\\\\\,

~\\\\\~

/ / / / / / ~ ,',,,\\\\,

r/ / / I \ \ \ LS1 -

Figure

LSI+

LS1 -

5

The expression o f t h e LS1 e p l t o p e from juveni le and adult L.stagnalls to the phagocytic capacity (phag)

on

hemocytes in relation of the Cells.

It is remarkable that in Juvenile L.stagnalls a larger percentage of the hemocytes displays t h e LS1 e p l t o p e than in adults. C a . 39% of the circulating hemocytes from juvenile is LSI+, whereas for adults this percentage is ca. 14%. T h i s s e e m s to imply that the number of LSI+ hemocytes decreases as the snal Is mature. As, however, the number of circulating hemocytes per pl hemolymph is higher for adult snails and as also their blood volume i s much larger in comparison to juveniles (cf, Dikkeboom et al. 1984), the LSI+ population is in fact expanding during snail maturation. The fact that the majority of the LSI+ population shows prol i ferative activity is not surprising, therefore. The increase of the proportion of LSIhemocytes during snai I maturation is even higher than that of LSI+ cel Is. This cannot be explained by a high prol iferative activity of LS1hemocytes in the circulation, a s we f o u n d low percentages of LSl-hemocytes showing incorporation of bromodeoxyuridine. There might be several explanations for this apparent contradiction. Firstly, LSI+ hemocytes might loose the epitope and become LSI-, i.e. t h e LS1 epitope might be a differentiation antigen. We h a v e , h o w e v e r , no indications for this hyPothesis. Another explanation might be that there is an influx -mainly of LSIhemocytesfrom the connective tissue into the circulation (cf. Sminla et al. 1983). The distribution of LSI+ and of LSI- cel Is in the connective tissue and their occurrence in a putative hemocyte producing organ (cf. Jeong et al. 1983) have, however, not been studied yet.

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100 RCA-

90 ~uvenile

adult

80 RCA--

_0

&

7G 60

E -5

50 ~0 30

RCA+

20 RCA+

~

I0 0 LS1 +

LS1 +

LS1 -

Figure

RCA+ LS1 -

6

The expression o f t h e LS1 e p i t o p e on hemocytes from juveni le and adult L.stagnalls in relation to the presence on the cells of surface carbohydrates recognized by Ricinus communls a g g l u t l n l n (RCA).

The morphology of the LSI+ hemocytes In b o t h age groups is very similar; in general they are small cells with a typical Iobopod, whereas in adults occasionally small cells with branched pseudopods were found. It is striking that of the percentage of hemocytes expressing t h e LS1 e p l t o p e a smaller part contains the enzyme peroxldase as compared to LSIhemocytes. LSI+ hemocytes are also phagocytical ly less active than LSIcells. The morphological and functional characteristics together with the high proliferative activity of the LSI+ hemocytes make it conceivable that these cells are the so-called round hemocytes which constitute a b o u t 70% o f t h e t o t a l juvenile and about 12% o f the total adult hemocyte population (Dlkkeboom et al. 1984; 1985b). It is surprising that in adult snails virtually all round cel Is are LSI+, whereas in juveni le snai Is only about half of the round hemocytes express t h e LS1 e p l t o p e . Thus, a round hemocyte is not necessarily LSI+. The results of our study are comparable with those of Yoshlno and Granath (1983; 1985). Using monoclonal antibodies raised against hemocytes of B.glabrata they Identified antigenlcal ly distinct hemocyte subpopulations in this snail species. BGHI+ hemocytes are morphological ly dlstlnghulsable from the BGHI- cells and, moreover, BGHI+ cells show a lower phagocytic activity and lysosomal enzyme (acid phosphatase) content than do BGHI- cells. They too found a decrease in the proportion of hemocytes possessing the epatope as snails mature. Thus, BGHI+ cells share morphological and functional characteristics with LSI+ hemocytes of L.stagnalls. Staining of B.glabrata hemocytes with moAb LS1 gave negatlye results, however (R. Dlkkeboom, unpublished observations). For Investigations on the possibility of species specificity of snail hemocyte membrane determinants further studies on crossreactlvity of monoclonal antibodies directed against hemocytes of L.stagnalls and B.glabrata are needed.

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100 90

STA+

80 2

juvenile

aduH

70 STA+

o 0 o E

50 40 STA+

2O I 0

STA-

STA+

0 LS1 +

LS1 -

LS1 +

LS 1

-

Figure 7 The expression o f t h e LS1 e p i t o p e on hemocytes from juveni le and adult L.stagnalls in relation to the presence on t h e c e l l s of surface carbohydrates recognized by Solanum tuberosum agglutinin (STA).

Results of the staining with 13 d i f f e r e n t lectlns showed that six of them did not react with the hemocytes at all, five bound to all snal I cells and two lectins recognized carbohydrates present on t h e m e m b r a n e o f o n l y a part of the hemocyte population. The fact that the lectins BS-IA4, BS-IB4, DBA, ECA, PNA a n d SBA do not bind to L.stagnalis hemocytes could have several reasons. The most obvious one is the absence from the hemocyte glycocalyx of the carbohydrate residues recognized by these lectins. In other studies on b i n d i n g of lectins to snail hemocytes comparable (negative) results have been found; the lectlns BSA, DBA a n d SBA were found not to react with hemocytes of B.glabrata (Schoenberg and Cheng 1980; Yoshino 1983) and not with those of Helix pomatla (Renwrantz and Cheng 1977). Another explanation might be t h a t the sugar residues, although present on the membrane, are not accessible for the lectins because of e.g. sterical hindrance and/or masking by o t h e r membrane molecules. Also, the fixatives used (i.e. formaldehyde and glutaraldehyde) might have Influenced lectin binding; moreover, the treatment of hemocytes with methanol/H202 to eliminate endogenous peroxidase m ght cause loss of carbohydrate residues associated with glycol Ip ds (cf. Alroy et al. 1984). The lectins APA, C o n A, LCA, LTA a n d WGA r e a c t e d w t h 100% o f t h e hemocytes of both age groups. Specific inhibition of lectln binding by the appropriate sugars indicates the presence of galactosyl , glucosyl , mannosyl , fucosyl and N-acetyl-glucosamlnyl residues on the hemocyte membranes. There seems to be no noticeable modification of the carbohydrate composition during hemocyte maturation, as the five lectins show simi lar binding to hemocytes of juveni le and adult snai Is.

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LSI+ HEMCOYTES

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• i~

~

Figure 8 Fluorescence of hemocytes from adult L.stagnalis incubated with Solanum tuberosum agglutinin (STA; conjugated with F I T C ) a n d moAb LS1 (visualized with a TRITC conjugate). Figure 8a s h o w s STA+ h e m o c y t e s , arrows point at hemocytes that are also positive f o r t h e moAb LS1 a s c a n b e s e e n i n 8 b . N nucleus; X 1000.

The present results o f t h e Con A s t a i n i n g are in line with those presented previously on adult L.stagnalis (Sminia et al. 1981) and comparable with those obtained for B.glabrata hemocytes (Yoshino 1981). Our results o n WGA b i n d i n g are in contrast with Yoshino's report (1983) that this lectin does not bind to surface determinants of B.glabrata hemocytes. This might be due to differences in the snal I species and/or experimental conditions. Binding of the two lectins RCA a n d STA to L.stagnalis hemocytes indicates the presence of ~-D-galactosyl and N-acetyl glucosaminyl groups on the surface membrane. The reduction In the percentage of RCA+ h e m o c y t e s w h e n t h e s n a i I s m a t u r e c o u l d be due to a relatively larger supply of RCAcells by proliferation and/or migration from the connective tissue or putative hemocyte producing organ; further studies on the distribution in these tissues of hemocytes reacting with lectins might shed more I ight on this problem. The proportion of STA+ hemocytes is only si ightly different between the two age groups. The LSIpopulation of both juveni le and adults is consistently negative for RCA. This could lead to. the speculation that t h e LS1 e p l t o p e is a glycoconjugate in which ~-D-galactosyl residues are present. However, the LSI+ population is not entirely RCA+, b u t h e t e r o geneous: b o t h RCA+ a n d RCA- c e l I s a r e f o u n d .

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With STA the opposite seems to be the case: in the LS1population both STA+ a n d STA- cells can be found, but virtually none of the LSI+ hemocytes reacts with this lectln. Moreover, the STA+ and RCA+ hemocyte subpopulatlons are not complementary; double labelling experiments using both lectlns showed that the subpopulatlons partly overlap each other (R. Dlkkeboom, not published). Thus, the presence of ~-D-galactose does not exclude the presence of N-acetyl-glucosamine and vice versa; furthermore, our results Indicate that there is no positive correlation between presence or absence of certain carbohydrates and the occurrence o f t h e LS1 e p i t o p e . In conclusion, we h a v e s t u d i e d proliferative activity, phagocytic capacity, lysosomal peroxldase content and lectln binding of the moAb LS1 r e c o g n i z e d hemocyte subpopulatlons of Juvenile and adult L.stagnalls. In both age groups, the LS1 e p l t o p e Is a surface membrane marker of less differentiated hemocytes. Future studies are needed on the ultrastructure of the LSI+ hemocytes, the possible function of the LS1 epltope and its biochemical characterization. Moreover, moAb LS1 (and other moAb's) and lectlns should be applied to isolate hemocyte subpopulatlons In order to further characterize them morphologically and functionally.

ACKNOWLEDGEMENTS The

authors

wish

to

thank

Dr. Taede Smlnla the manuscript.

for

critical

ly

reading

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Yoshino TP (1981) Comparison of concanaval determinants on hemocytes of two Blomphalarla stocks: receptor binding and redistribution. D e v Comp I m m u n o l 5 : 2 2 9 - 2 3 9 . Yoshino TP mol l u s c a n D e v Comp

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TP,

Granath J r WO ( 1 9 8 5 ) Surface antigens (gastropoda) hemocytes: functional cell subpopulations recognized by a monoclonal J Invertebr Pathol 45:174-186.

glabrata

Received: August, 1987 Accepted: September, 1987

of Biomphalaria heterogeneity In antibody.