Histochemical Journal 22, 595-603 (1990)

Hydrolytic enzymes associated with the granular haemocytes of the marine mussel Mytilus edulis R. K. P I P E Natural Environment Research Council, Plymouth Marine Laboratory, Citadel Hill, Plymouth, PL1 2PB, UK Received 14 March 1990 and in revised form 27 April 1990

Summary The ultrastructural localization of a range of hydrolytic enzymes has been investigated in the granular haemocytes of the marine mussel Mytilus edulis. Arylsulphatase activity and immunocytochemicallocalization of ~-glucuronidase and elastase were demonstrated within the large granules of the haemocytes. Lysozyme and cathepsin B were both localized within all sizes of granule, however, at high dilutions the primary antibody against lysozyme was also restricted to the large granules. The labelling density for cathepsin B antibody tended to be very low. Antibodies for cathepsin G showed a clear, discrete labelling which was restricted to the granules of haemocytes containing small granules. The fact that antibodies raised against human proteinases recognize invertebrate enzymes suggests that there must be a certain degree of structural similarity between the human proteinases and the enzymes present in the mussel haernocytes indicating either convergence or conservation of the enzyme molecules. The presence of a range of hydrolytic enzymes including proteinases, glycosidases and sulphatases within the large granules shows that these granules are a form of lysosome. The reduction in activity of lysosomal enzymes in haemocytes following adhesion to glass is evidence for release of the enzymes from the granules (degranulation). The possibility of a serine protease being specifically associated with the small granules and its role as a cytolysin are discussed.

Introduction

Circulating haemocytes play a key role in the immune response of molluscs. The cells are capable of recognition, with the aid of lectins, and possible chemotactic migration towards invading pathogens (Cheng & Howland, 1979), followed by attachment and finally endocytosis of the pathogen. An integral part of this progress of phagocytosis involves the release of cytotoxic agents as a means of killing the invading pathogen. Two basic mechanisms for cell killing have been described for molluscan haemocytes. The first comprises the respiratory burst with the release of highly reactive oxygen metabolites including superoxide, hydrogen peroxide, singlet oxygen and hydroxyl radicals (Nakamura et al., 1985; Dikkeboom et al., 1988). The second involves the release of lysosomal enzymes or other lysins from the haemocytes (Cheng et aI., 1975; Cheng & Yoshino, 1976a,b; Foley & Cheng, 1977; Wittke & Renwrantz, 1984; Cheng & Mohandas, 1985; Leippe & Renwrantz, 1985, 1988; Mohandas & Cheng, 1985; Mohandas et al., 1985). The release of lysosomal enzymes by granular haemocytes of bivalve molluscs is accompanied by degranulation of the cell (Foley & Cheng, 1977). Studies using enzyme cytochemistry have demons0018-2214/90 $03.00 +.12 9 1990 Chapman and Hall Ltd.

trated certain lysosomal enzymes associated with the granules (Fankboner, 1971; Feng et al., 1971; Yoshino & Cheng, 1976; Moore & Lowe, 1977; Bayne et al., 1979), however, only acid phosphatase has been localized at the ultrastructural level (Fankboner, 1971; Yoshino & Cheng, 1976). The circulating haemocytes of Mytilus eduIis have been classified into two morphologically distinct types, granular and agranular cells (Moore & Lowe, 1977; Cheng, 1981; Rasmussen et al., 1985; Pipe, 1990). The granular haemocytes have been shown recently to be heterogeneous in terms of the lectin binding characteristics of their granules (Pipe, 1990). The present work was undertaken in order to determine the hydrolytic enzyme content of the granutes as a preliminary study prior to investigating the functioning of the different granules, in terms of the release of cytolytic agents, during phagocytosis. The localization of a range of hydrolytic enzymes was studied using standard cytochemica! methods and immunocytochemical techniques employing commercially available antibodies. In addition, the effect of degranulation on cytochemically measured lysosomal enzyme activity within the haemocytes was investigated.

596

Materials and methods

Animals Marine mussels, M. edulis (50-70ram shell length) were collected at low tide from Sharrow Point, Whitsand Bay, Cornwall, England. Haemocytes were extracted on the day of collection from the posterior adductor muscle with a sterile 5 ml syringe.

Cytochemistry A fixed haemocyte suspension was prepared by extracting 0.5 ml of haemolymph into a 5 ml syringe containing I ml fixative (0.1M PIPES-buffered 2% formaldehyde freshly prepared from paraformaldehyde and 2.5% glutaraldehyde, pH 7.4, containing 2mM CaCI2, total osmolality 1000 mOsm). Fixation was for 30 min after which the haemocyte suspensi6n~ pooled from 5 animals, was given 2 rinses in 0.1 M acetate buffer, pH 5.5, and incubated for arylsulphatase activity using the barium precipitation method (HopsuHavu et al., 1967; Hopsu-Havu & Helminen, 1974) with slight modification (Pipe & Moore, 1985, 1986; Pipe & Da Silveira, 1989). Briefly, the haemocytes were incubated at 37~C for 3 h in a medium containing p-nitrocatechol sulphate as the substrate and barium (as BaC12)as the capturing ion in 0.1 M acetate buffer, pH 5.5. Controls were carried out by. omitting the substrate from the incubation medium. Following incubation, the cells were given 2 rinses in 0.1 M acetate buffer, encapsulated in 10% bovine serum albumen (BSA) (Pipe, 1990) and post-fixed in 1% osmium tetroxide in 0.1 M cacodylate buffer for Ih. The BSA-gel containing the cells was then rinsed in cacodylate buffer, dehydrated in an ethanol series and processed through propylene oxide into Taab epoxy-resin. Ultrathin sections were cut on a Reichert Ultracut E ultramicrotome and examined, without counterstaining, in a Philips 300 electron microscope.

Immunocytochemstry Haemolymph (0.5 ml) was collected into I ml fixative and left to fix for 30 rain at room temperature. After fixation, the cells were given 4 washes in 0.2 M Tris buffer, pH 7.8, 850 mOsm, encapsulated in BSA and processed through an ethanol series into LR white resin. Following overnight infiltration the samples were embedded in gelatin capsules and polymerized at 50~ for 20 h. Sections were collected on nickel grids and pre-incubated in phosphate-buffered saline (PBS) for 5 rain prior to incubation in the primary antibody. The following antibodies were investigated: (a) antielastase, (b) anti-cathepsin B, (c) anti-cathepsin G, (d) antilysozyme, all raised in sheep, against human antigens (The Binding Site, Birmingham, UK), and (e) anti-~glucuronidase raised in rabbit against antigen from Escherichia coli (5 Prime 3 Prime Inc., Pennsylvania, USA). A range of dilutions in PBS pH 7.2, between 1 : 10 and 1 : 1000 and incubation times between 2 and 4.5 h were tested for each of the primary antibodies. Incubations were carried out at room temperature within a humidity chamber. Following incubation in primary antibody the sections were washed thoroughly in PBS and incubated in gold-labelled secondary antibody. Gold (15nm) conjugated goat anti-rabbit IgG (BioCell, Cardiff, UK) was used as the secondary antibody for ~-glucuronidase and gold (10nm) conjugated donkey anti-sheep IgG (BioCell, Cardiff, UK) was used as the

PIPE secondary antibody for all other antigens: Again, a range of dilutions in PBS (containing 0.5% BSA and 0.05% Tween 20) and incubation times were investigated. Controls were carried out by omitting the primary antibody and replacing it with non-immune serum and buffer, in addition, the primary antibody was incubated with its antigen (Sigma Chemical Co.) prior to the immuno-labelling. The sections were rinsed thoroughly in PBS with a final wash in distilled water and examined, without counterstaining in a Philips 300 electron microscope.

Effect of degranulation on lysosomal enzyme activity Haemolymph (0.5 ml) was collected into sterile 5 ml syringes containing 0.5 ml 0.2 M Tris-buffered saline (TBS), pH 7.8, 850 mOsm; a single drop of haemolymph was used to produce haemocyte monolayers as described previously (Pipe, 1990) following essentially the method of Renwrantz et al. (1985). Briefly a single drop of haemolyrnph was placed at the end of a glass microscope slide and left in a moisture chamber for 5 rain to allow the cells to attach. Excess cells were washed off with TBS before rounding up the attached haemocytes by incubation in TBS for 20rain at room temperature. The monolayers were then fixed for 30 rain in fixative. The haemolymph not used for producing monolayers was added to i ml of fixative and also left to fix for 30min. It has been shown previously using electron microscopy (Pipe, 1990) that the formation of monolayers causes degranulation of the haemocytes which does not occur in haemocyte suspensions. Following fixation, the haemocyte monolayers and suspensions were washed in TBS with a final rinse in 0.1M acetate buffer pH 5.5. The haemocytes were then incubated for f~-glucuronidase and arylsulphatase activity using the naphthol-AS-BI method (Pearse, 1972) and simultaneous coupling with the diazonium salt Fast Violet B (Sigma Chemical Co.). Incubations were overnight (17h) at room temperature. Following incubation the cells were rinsed in acetate buffer and optical density readings taken on individual cells using a Vickers M85 scanning microdensitometer at a wavelength of 560 nm with a spot size'of 0.5 ~m diameter, a mask size A3 and a x 40 objective.

Results An electron d e n s e precipitate of barium sulphate indicating arylsulphatase activity was localized within the granules of the h a e m o c y t e s (Fig. 1). Previously the granular haemocytes, which are the major constituent of the blood cell p o p u l a t i o n of M. edulis, have b e e n classified into those containing large (0.5-1.5~m) granules and those containing small (0.2-0.3~m) granules (Pipe, 1990). The arylsulphatase activity localized in the p r e s e n t s t u d y was invariably associated with the granular h a e m o c y t e s containing large granules. Relatively few granules of each h a e m o c y t e d e m o n s t r a t e d arylsulphatase activity and in m a n y cases only a part of the granule contained reaction product. The controls, incubated w i t h o u t substrate, did not s h o w any reaction product. The i m m u n o c y t o c h e m i s t r y gave positive results for

Hydrolytic e n z y m e s in mussel h a e m o c y t e s

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Figs 1-3. Unstained thin sections of granular haemocytes from Mytilus edulis demonstrating localization of hydrolytic enzymes. Fig. 1. Electron-dense barium sulphate reaction product showing acvitity for arylsulphatase x27000. Scale-bar 0.5 Fin. Fig. 2. Section incubated with anti-~-glucuronidase (raised in rabbit) followed by 15nm goldqabelled goat anti-rabbit • Scale-bar 0.5 ~m. Fig. 3. Section incubated with anti-elastase (raised in sheep) followed by 10 nm gold-labelled donkey anti-sheep • 600. Scale-bar 0.2 ~m.

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Figs 4 and 5. Unstained thin sections of granular haemocytes from Mytilus r incubated with antiqysozyme (raised in sheep) followed by 10 nm goldqabelIed donkey anti-sheep. Fig. 4. Localization with low dilution (1:100) of primary antibody x58 000. Scale-bar 0.2 ~rn. Fig. 5. Localization with high dilution (1 : 1O00) of primary antibody x72600. Scale-bar 0.2 pan.

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Fig. 6. Unstained thin section of granular haemocyte from Mytilus edulis incubated with anti-cathepsin B (raised in sheep) followed by 10 nm gold-labelled donkey anti-sheep x34 700. Scale-bar 0.5 ~m. Fig. 7. Unstained thin section of granular haemocyte from Mytilus edulis incubated with anti-cathepsin G (raised in sheep) followed by 10 nm gold-labelled donkey anti-sheep x88 500. Scale-bar 0.2 gm.

600 all 5 antibodies investigated. In all cases controls incubated without primary antibody were completely negative, and those with the primary antibody preincubated with the antigen showed a reduction in immunolabelling. The immunocytochemical localization of ~glucuronidase demonstrated the enzyme within the haemocytes containing large granules and generally restricted to the largest granules (Fig. 2). The colloidal gold labelled antibody showed a quite dense and even distribution throughout the matrix of the granules with minimal background staining elsewhere in the haemocytes. The antibodies for elastase showed a very similar distribution to ~-glucuronidase, this also being localized within the large granules with an even distribution throughout the granules (Fig. 3). The degree of labelling was less than for ~-glucuronidase and again the background staining was minimal. The antibodies for lysozyme and cathepsin B were both localized within all sizes of granule, however, the degree of labelling was quite different for the two antibodies. At low dilutions of lysozyme antibody, up to 1 : 100, the gold was located in all sizes of granule but with fairly high background label (Fig. 4). With dilutions of 1 : 1000 the gold was mainly restricted to the large granules with minimal background label (Fig. 5). The cathepsin B antibody was localized within all sizes of granule but generally at a low level of labelling (Fig. 6). The antibody for cathepsin G showed a very good discrete labelling which was restricted to the granules of the haemocytes containing small granules (Fig. 7). The experiments to investigate the effect of degranulation on lysosomal enzyme activity showed a significant decrease in enzyme activity when the haemocytes were prepared as monolayers (Fig. 8). Activity for [~-glucuronidase showed the most dramatic decline; the monolayer demonstrated only approximately a third of the reaction product when compared with the haemocyte suspension. Arylsulphatase activity in the monolayers resulted in approximately two thirds of the reaction product produced by the cell suspension. Discussion

The granules within the granular haemocytes of M. edulis clearly contain a number of hydrolytic enzymes. The results support previous findings, using lectin binding characteristics (Pipe, 1990), that the granules are heterogeneous in nature. The immunocytochemistry of the proteolytic enzymes, which employed antibodies raised to human antigens, demonstrates a certain degree of structural similarity between the human proteinases and the enzymes present in the haemocyte granules, suggesting either a considerable conservation or convergence of the enzyme mole-

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Fig. 8. Effect of degranulation of haemocytes on cytochemically determined lysosomal enzyme activity. Values are means from 5 animals with 20 readings per animal + 2 SE.

cules. Homology has been reported for a number of proteases isolated from micro-organisms and mammalian sources including viruses and retroviruses (Pearl & Taylor, 1987; Bazan & Fletterick, 1988), bacteria (Whitaker & Roy, 1967), yeast (Galjart et al., 1988) and fungi (Tang, 1979). Arylsulphatase, ~-glucuronidase and lysozyme are all lysosomal enzymes which are widely distributed in both vertebrate and invertebrate tissues. Lysozyme and O-glucuronidase have been reported previously in granular haemocytes of bivalve molluscs (Moore & Lowe, 1977; Bayne et al., 1979; Cheng & Downs, 1988); their ability to hydrolyze mucopolysaccharides (which are constituents of bacterial cell walls) together with enhancement of serum levels following bacterial challenge has led to the theory that they serve as nonspecific, humoral defence molecules (Cheng, 1976; Cheng et al., 1977). Arylsulphatase has been demonstrated in various cell types of M. edulis (Pipe & Moore, 1985; Pipe, 1987; Pipe da Silveira, 1989) but is predominantly associated with the secondary lysosomes of the digestive cells within the digestive gland (Pipe & Moore, 1986). In mammals arylsulphatase activity is localized within the granules of the blood phagocytes including eosinophils and monocytes (Bainton et al., 1976). Elastase is a serine proteinase which is localized in the azurphilic granules of human neutrophils, and is active at neutral pH unlike the lysosomal enzymes which have optimal activity at weakly acidic pH. Elastase degrades a wide range of connective tissue proteins and can lyse bacterial cell walls indicating a

Hydrolytic enzymes in mussel haemocytes bactereolytic function in leukocytes (Havemann & Janoff, 1978). Elastase activity has been reported in pathogenic free-living amoebae (Ferrante & Bates, 1988), however, there is a high degree of similarity between elastase and other serine proteinases including trypsin and chymotrypsin (Hartley, 1970) both of which have been reported previously in bivalves (Reid & Rauchert, 1970). Cathepsin B is a lysosomal cysteine proteinase which is synthesized by mammalian macrophages and is involved in intracellular protein degradation. There have been several reports of cathepsin B-like activity in bivalve molluscs, generally associated with the digestive gland (Reid & Rauchert, 1976; Chen & Zall, 1986; Zeef & Dennison, 1988). The enzymes discussed so far were all localized within the larger granules of the granular haemocytes, and included proteinases, glycosidases and sulphatase, demonstrating that these granules are a form of lysosome. The attachment of haemocytes to glass slides has been shown previously, by electron microscopy, to result in degranulation (Pipe, 1990); the present results, showing reduction in activity of lysosomal enzymes in attached haemocytes, indicates that degranulation is as a result of release of lysosomal enzymes from the granules. Previous studies by Cheng and co-workers have demonstrated degranulation and enzyme release in molluscan haemocytes following bacterial challenge (Cheng & Yoshino, 1976a,b; Foley & Cheng, 1977; Cheng et al., 1978; Cheng & Mohandas, 1985; Mohandas & Cheng, 1985; Mohandas et al., 1985). It is interesting that the small granules, which have been shown previously to be Helix pomatia lectin positive (Pipe, 1990), seem to contain only protease enzymes. Cathepsin G antibodies in particular showed a high affinity for these granules. Cathepsin G is a cationic neutral serine proteinase which is found in the azurphilic granules of polymorphonuclear leukocytes and shows distinct homology with chymotrypsin (Barrett, 1981). The enzyme exhibits potent in vitro antimicrobial activity which is believed to be nonenzymatic, resulting instead from the cationic nature of the molecule (Shafer et al., 1986). Antibodies to cathepsin G were the only antibodies to be localized solely in the small granules of the granular haemocytes, indicating that a serine protease is associated uniquely with these granules. Serine proteases have been shown to be a major protein component of cytotoxic lymphocyte granules in mammaIs. Their physiological functions are not known, but a role in cell mediated cytotoxicity is likely (Jenne & Tschopp, 1988). These so-called granzymes show a primary structure which is closely related to cathepsin G (Salvesen et al., 1987), and in some cases show the same glycosylation sites. It is tempting to speculate on the possibility that Helix pomatia lectin binds to the carbohydrate component of

601 a serine protease in the haemocytes and to further speculate on the likelihood of this being the cytolysin described previously in M. edulis (Whittke & Renwrantz, 1984; Leippe & Renwrantz, 1985, 1988). Clearly, isolation of the small granules is a necessary pre-requisite before purification and characterization of proteases associated with the granules can be carried out. Affinity chromatography with bound lectin from Helix pomatia appears to be a promising possibility for such a purification.

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Hydrolytic enzymes associated with the granular haemocytes of the marine mussel Mytilus edulis.

The ultrastructural localization of a range of hydrolytic enzymes has been investigated in the granular haemocytes of the marine mussel Mytilus edulis...
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