Histochemical Journal 21, 419-424 (1989)

Arylsulphatase cells

in echinoderm immunocompetent

C A L O G E R O C A N I C A T T I and A N N A M I G L I E T T A

Department of Biology, University of Lecce, Via Prov. Ie Lecce Monteroni 73100 Lecce, Italy

Received 30 August 1988 and in revised form 25 November 1988

Summary

Two peaks of arylsulphatase activity were detected biochemically in coelomocyte lysate preparations of seven different Echinodermata species. Both peaks were inactivated by sulphite and sulphate ions, indicating that Type II arylsulpha- tase is present in the coelomocytes of the species tested. Arylsulphatase was localized histochemically in the granules of spherula cells, suggesting that in echinoderms a common cell type with granulocyte-like functions is present. The enzyme was also localized in the amoebocytes of echinoid species.

Introduction

The reactions of invertebrates to experimentally or naturally introduced 'non-self' organisms and mole- cules are primarily of a cellular nature. In Holothuria polii (Holothuroidea), a series of cellular events, of which phagocytosis appears to be the principal one, occurs when a single dose of formalinized sheep red blood cells is injected into the wide coelomic cavity (Canicatti & D'Ancona, 1988). The phagocytosed materials are degraded intracellularly, probably by lysosomal enzymes that, as in other invertebrates (Cheng & Cali, 1974; Cheng, 1983, 1986), could be released into secondary phagosomes.

Very little information has been acquired on the role of lysosomal enzymes during the cellular response of H. polii. Canicatti (1988) found, by using arylsulpha- tase as a marker of lysosomal activity, that this enzyme is present in the granules of circulating spherula cells. These cells are also present in large numbers in structures (the brown bodies) that actively participate in digestion and clearance of natural and experimental foreign material introduced into the holothuroid body (Canicatti & D'Ancona, 1988).

Arylsulphatase are lysosomal hydrolases that catalyse the hydrolysis of sulphate bonds of a variety of synthetic substrates including p-nitrocatecholsul- phate (Dodgson & Spencer, 1957). They are known to play a role in granulocyte functions, including in- flammatory phenomena (Wasserman et al., 1975). They have been cytochemically localized in mast cells (Orange & Moore, 1976), basophils (Baker & Bainton, 1980) and, in a latent form, in neutrophils (Bainton & Farquhar, 1968) and eosinophils (Bainton & Farquhar,

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1970). In H. polii two pH optima for arylsulphatase activity in cell lysate preparations have been found, at pH 5.0 and pH 5.8. Both increased after injection of zymosan particles or formalinized sheep erythrocytes, indicating a probable engagement of the enzyme during phagocytosis of particulate materials. Sulphite and sulphate ions inhibited the activity of the enzyme indicating that Type II arylsulphatase is present in H. polii coelmocytes (Canicatti, 1988). No data are avail- able on the presence of these enzymes in coelomocytes of echinoderms other than H. polii.

The echinoderm coelom0cytes appear to fall into six main categories: phagocytic amoebocytes, spherula cells, vibratile cells, haemocytes, crystal cells and progenitor cells (Smith, 1981). This coelomocyte pro- file can vary considerably not only between species but also between individuals of the same species. However, spherula cells appear to be ubiquitous in the Echinodermata phylum (Smith, 1981). Since in H. polii the localization of arylsulphatase activity in spherula cells could indicate a granulocytic-like function for this cell type, it is useful to ascertain if this enzyme could also represent a marker characterizing a functionally common cell type in echinoderms.

In the present paper biochemical and cytochemical evidence is produced on the presence of arylsulpha- tase activity in coelomocytes of different Echi- nodermata species.

Materials and methods Uniformly shaped adult Marthasterias glacialis and Echinaster sepositus (Rety.) (Asteroidea), Paracentrotus lividus (Lam.),

420 CANICATTI and MIGLIETTA

Arbacia lixula (L.) and Spherechinus granularis (Lam.) (Echin- oidea), Holothuria tubulosa (Gm.) and Holothuria forskali (Delle Chiaje) (Holothuroidea) were collected from Porto Cesareo, Lecce. The coelomic fluid was obtained by transversal incision of the arms for the asteroid species, by cutting a slit in the peristomial membrane for the echinoid species, and by longitudinal incision of the wide coelomic cavity for the holothuroid species. In all cases the coelomic fluid was collected in anticoagulant containing 0.01 M EDTA and 0.3 M Hepes in 0.5 M NaCI (Hepes-NaC1) to prevent clot formation (Bertheussen & Seljelid, 1978). The coelomocytes were pelleted by centrifugation at 400g for 15min at 4°C, resuspended in 2ml of distilled water and subjected to sonication in a Branson Sonifer model 15B at 50% duty cycles at 0°C. When complete lysis was obtained (4min), the sonicate was centrifuged at 12000g. The supernatant was separated and used to estimate total protein content according to Lowry's (1951) method.

Solutions of coelomocyte lysate with a protein content of 0.14mgml 1 were prepared and arylsulphatase activity was biochemically determined according to Franklin et al. (1983). Briefly, 20~tl of coelomocyte lysate samples at constant protein concentration were mixed with 80~1 of 6.2mM p-nitrocatechol sulphate (Sigma) in 0.2M acetate buffer at different pH values (4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8 and 6.0). Incubation took place in a water bath at 37 ° C for I h and was stopped by addition of 100 ~tl of 5 M NaOH. The orange colour developed was spectrophotometrically read at 515 nm.

Inactivation of the enzyme activity was effected by preincubating 20 ~1 of coelomocytes lysate samples at 56 ° C or by adding 80 ~1 of 12.5mM sodium sulphate (Na2SO4) or sodium sulphite (NaRSO3).

Arylsulphatase activity in Echinodermata coelomocytes was localized by using Goldfischer's medium containing p-nitrocatechol sulphate as substrate (Goldfischer, 1956). The smears of coelomocytes were air-dried, fixed for 30 min in 1.5% glutaraldehyde in 0.1M sodium cacodylate buffer (pH 7.4) containing 1% sucrose, washed three times in acetate buffer (pH 5.7) and then incubated in the dark at 30°C for 2 h with Goldfischer's medium. After incubation, the cells were washed five times for 5 min in acetate buffer (pH 5.0) supplemented with 7% sucrose. The reaction product was converted by incubation in 2% ammonium sulphide in acetate buffer (pH 5.4) for 10 min and, after several washes, examined under a light microscope. Control preparations were incubated without substrate.

R e s u l t s

Biochemical determination of arylsulphatase Coelomocyte lysates of all seven Echinodermata spe- cies examined possessed arylsulphatase. W h e n the e n z y m e activity was tes ted at different p H values, two major peaks were detectable in M. glacialis and E. sepositus (Asteroidea) and in H. tubulosa and H. forskali (Holothuroidea) . The higher activity was at pH5 .0 , t h e lower one at p H 5.8. The echinoids P. lividus and A. lixula s h o w e d two peaks too: the h igher activity, howeve r , was at p H 5.8 ra ther than at p H 5.0. In S. granularis the ary lsu lphatase activity was detectable

only at p H 5.0. In some individuals of M. glacialis and E. sepositus and, to a minor extent, in all ech inoderm coelomocyte prepara t ions , variat ions in the higher peak were found. It was, in fact, located at p H 5.2 ra ther than at p H 5.0. The arylsulphatase activity of the coelomocytes d e p e n d e d on the total prote in content of the lysate prepara t ions and, consequent ly , on the n u m b e r of cells p resen t in each animal. Howeve r , as s h o w n in Fig. 1, each animal had, at the same prote in concentra t ion (0.14 m g ml 1), a different absorbance peak. A. lixula was, a m o n g the Echi- noidea, the mos t reactive species; the least reactive one was P. lividus. M. glacialis and E. sepositus (Asteroidea) did not show significant differences. The sea cucumber H. tubulosa was more reactive than H. forskali.

In all cases the e n z y m e activity was s t rongly inhibi ted by 12.5 mM solutions of sod ium sulphite and sod ium sulphate. Moreover , thermal inactivation was demons t r a t ed at 56 °C.

Cytochemistry Cytochemical evidence for the presence of arylsulpha- tase in Echinodermata coelomocytes was obta ined us ing Goldf ischer ' s me thod . After incubat ion of the cells wi th Goldfischer 's med ium, the dense reaction product , lead sulphite, was localized mainly in the

¢U u) _.> O

O O 1.0- E o

o 0 7

E o'J

E '¢ 0.5- ei

L .

E i,n

d AI pI I SIg

SPECIES

t I

Mg Es

TESTED

I Ht

I H f

Fig. 1. Comparison of arylsulphatase activity present in coelomocyte lysate preparations (protein concentration 0.14 mg ml 1) of seven species of echinoderms. A1, Arbacia lixula; P1, Paracentrotus lividus; Mg, Marthasterias glacialis; Es, Echinaster sepositus; Ht, Holothuria tubulosa; Hf, Holothuria forskali. The vertical bars represent one S.E.

Arylsulphatase in echinoderms

granules of the spherula ceils in the seven species examined (Fig. 2). However, differences in the inten- sity of reaction were observed. As shown in Table 1, a strong reaction was observed in spherula cells of A. lixula, whereas E. sepositus and S. granularis spherula cells were moderately positive. A weak reaction was observed for P. lividus, M. glacialis, H. tubulosa and H. forskali positive cells.

421

In some preparations, a feeble reaction (Table 1) was produced by amoebocytes of the echinoid species A. lixula, P. lividus and S. granularis. It was mainly localized in granules (Fig. 3), probably lysosomes, randomly distributed in the cytoplasm. Amoebocytes of the remaining species examined produced no positive reaction.

!

Fig. 2. Phbtomicrograph of echinoderm arylsulphatase-positive spherula cells. (a) M. glacialis; (b) E. sepositus; (c) S. granularis; (d) P. lividus; (e) A lixula; (f) H. tubulosa; (g) H. forskali. No counterstaining, x 1250.

422 CANICATTI and MIGLIETTA

Table 1. Cytochemical activity of arylsulphatase in positive coelomocytes of different echinoderm species.

Arylsulphatase activity

Species Amoebocytes Spherula cells

Asteroidea Marthasterias glacialis - + Echinaster sepositus - + +

Echinoidea Paracentrotus lividus + + Arbacia lixula + + + + Spherechinus granularis + + +

Holothuroidea Holothuria tubulosa - + Holothuria forskali - +

the

+ + +, strong reaction; + +, moderate reaction; +, weak reaction; +_, feeble reaction -, no reaction.

Discussion

The presence of lysosomal arylsulphatase has been shown in coelomocytes of Echinodermata. Two major peaks, at pH 5.0 and 5.8, can be measured biochemi- cally in all the species examined except S. granu lar i s ,

indicating that, as in H. polii (Canicatti, 1988), there are at least two different arylsulphatases. Their pH optima, however, differ from those of analogous enzymes present in mammalian mast cells, identified by Lynch et al. (1978) as arylsulphatases A and B, with pH optima 4.2 and 6.2 respectively. These differences could be a result of the species specificity of the enzymes.

As is well known, sulphatase B can clearly be distinguished from sulphatase A by its alkaline isoelectric point and its lower molecular weight; its substrate specificity, however, is approximately the

Fig. 3. Photomicrograph of (a) P. lividus, (b) S. granularis, (c) A. lixula arylsulphatase-positive amoebocytes. No counterstaining, x 1250.

Arylsulphatase in echinoderms 423

same as that of sulphatase A (Hopsan-Havu & Helminen, 1974). Sulphate and sulphite ions are inhibitors of arylsulphatase (Dodgson & Spencer, 1957). The specified inhibitory capability of these ions allows distinction between two principal types of arylsulphatase:Type I is not inhibited by sulphate ions whereas Type II, which includes arylsulphatases A and B, is inhibited (Hopsan-Havu & Helminen, 1974). In echinoderms there are not yet sufficient biochemical data on the presence of the two arylsul- phatase types. However, on the basis of distinct pH optima, it is reasonable to propose the presence of arylsulphatase A and B enzyme in echinoderm coelomocytes. Both are inactivated by sulphate and sulphite ions. Preincubation of coelomocyte lysate samples with 12.5mM Na2SO 3 or NaRSO 4 inhibited catalysis of the p-nitrocatechol sulphate used as substrate. This fact could be an indication that Type II arylsulphatases are present in echinoderm coelomo- cytes.

In all Echinodermata species examined the enzyme activity seems to depend on the protein content and consequently on the number of circulating cells, although at the same protein concentration, A. lixula has a higher arylsulphatase activity and H. forskali a lower activity. This could be due to different enzyme contents in the different species. However, we cannot exclude the possibility that a different physiological status of the aimals at the moment of collection contributed to the different reactivities observed.

Cytochemical evidence for the presence of arylsul- phatase in echinoderm coelomocytes was obtained using Goldfischer's medium. The positive cells in echinoderms were spherula cells. Although different degrees of reactivity could be observed among the species examined, the reaction was clearly localized in the spherical inclusions of the cells, indicating that these granules have a lysosomal origin. Spherula cells occur in echinoids (Boolootian & Giese, 1958), holothuroids (Hetzel, 1963; Fontaine & Lambert, 1977) and (described as spherical corpuscules) asteroids (Kanungo, 1984). The cells are round or ovoid having a diameter ranging from 8 to 20 ~tm; their nuclei are small, irregular and eccentrically placed. The cyto- plasm is filled typically with numerous large and spherical inclusions (Smith, 1981). There are insuf- ficient data to draw any definite conclusion about the function of these cell types. In some cases they have been associated with wound repair (Endean, 1966), in others they are observed to occur in large numbers during inflammatory response (Canicatti & D'An- cona, 1988). In H. polii the granules of spherula cells contain Type II arylsulphatase (Canicatti, 1988) and since spherula cells are present in large numbers in brown bodies (complex structures associated with elimination of unwanted materials (Canicatti & D'An- cona, 1988)), it is reasonable to propose a granulocyte-

like function for these cells. In the echinoderm species examined, spherula cells also contain arylsulphatase, and, if we assume this enzyme to be a marker of granulocyte-like functions, a common morphological and functional granulocyte-like cell type seems to be present in the Echinodermata phylum.

The feeble reaction observed in the amoebocytes of A. li'xula, P. lividus and S. granularis supports the idea of the possible presence of arylsulphatase enzymes in this Cell type also. Surprisingly no reaction was detectable in amoebocytes of the remaining species examined. However, the lack of reaction products in the amoebocyte site does not necessarily mean a lack of enzyme activity. As suggested for H. polii (Canicatti, 1988), it is possible that amoebocytes contain arylsul- phatase in a latent form.

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