Biol Cell (1992) 76, 97-102 © Elsevier, Paris

97

Original article

Adhesive properties of sea cucumber coelomocytes

Calogero Canicatti, Antonella Rizzo, Maria Rosa Montinari

Department o f Biology, University o f Lecce, 73100 Lecce, Italy (Received 13 April 1992; accepted 11 September 1992)

Summary - The adhesive properties of the coelomocytes of the sea cucumber, Holothuria pofii, have been investigated by studying their ability to attach to glass coverslips in vitro, and their morphology examined by scanning electron microscopy. Both amoebocytes and spherule cells in cell suspensions attached themselves to glass coverslips, but spreading activity was restricted to an amoebocyte subset which assumed an extremely flattened morphology. Coelomocyte adhesion was a time- and temperature-dependent phenome- non and required cations for attachment to the glass surface. Mg 2+ ions were more effective than Ca 2÷ in facilitating cell binding. The addition of potassium cyanide or sodium azide to the cell suspension did not inhibit amoebocyte attachment but vinblastine did. Cytochalasin B had no effect. Cell adhesiveness was greatly enhanced with both coelomic fluid and purified 220-kDa coeiomocyte- aggregating factor.

amoebocytes / adhesion / vinblastine / cytochalasin B / Hoiothuria polii (Echinodermata)

Introduct ion

Several cell-types, at all phylogenetic levels, are able to ad- here to each other to form aggregates for part icular pur- poses. In m a n y invertebrates specialized cells aggregate a round foreign material to fo rm encapsulat ing bodies in which the provoking agents are confined [1 -5 ] . During such an encapsulat ion response in Galleria mellonella, haemocytes secrete a substance which is believed to mediate adhesion of the cells to the foreign implant [6]. Recently a factor responsible for cell adhesion has been isolated f rom the crayfish Pacifastacus leniusculus [7]. It is believed to be released f rom degranulat ing haemocytes , and has a molecular mass o f about 76 kDa. This crayfish cell- adhesion factor also promotes encapsulat ion in vitro [8].

In the sea cucumber Holothuria polii, three major categories o f circulating cells have been described, namely, amoebocytes, spherule cells (types I - l i D , and progenitor cells [9]. Each cell-type is defined by its cytochemical staining properties [10] and, most probably, by a def'mite function. The cells are able to establish cell-cell contact, both during clotting [11] and encapsulation [5, 12]. Clotting is a three- stage event in which cells progressively aggregate into a large mass which then contracts to form the clot [11, 13]. Encap- sulating bodies, on the other hand, are complex scavenger structures consisting of a variable number o f nodules sur- rounding an internodular mass, and are large structures con- ventionally referred to as brown bodies [5, 12]. The nodules result f rom an aggregation o f layers o f amoebocytes which surround the foreign material [5]. In both clotting and en- capsulation it has been observed that the cell are connected to one another by their f'llipodia. This connection between participating ceils is not due to junctional complexes and, furthermore, no fusion occurs between reactant cells [5, 11]. Since adhesive interactions may be implicated in these events, the present study has been designed to explore the adhesive properties o f Holothuria polii circulating cells in vitro, and to determine the effects o f different substrata and various chemicals and metabolic inhibitors, in this process.

Materials and methods

Collection of animals and coelomocytes

Sea cucumbers, Holothuria polii (Echinodermata , Holothuroidea), were collected from Porto Cesareo (Lecce) and kept in aerated sea water. The coelomic fluid from each individual was obtained by making a longitudinal incision in the wide coe- lomic cavity. It was collected in an anticlotting medium (500 mM NaCI, 20 mM Tris-HCl, 30 mM EDTA, pH 7.5) in a 5:1 ratio of anti-coagulant and coelomic fluid, respectively. The coelom- ic of fluid was centrifuged immediately at 400 g for 5 min at 4°C. The pelleted cells were resuspended in Edds' isotonic salt solu- tion (EIS) prepared according to Edds [14]. Cell densities were determined using a hemocytometer.

Cell adhesion assay

Glass coverslips, rendered pyrogen-free by incubation at 180°C for 4 h, were coated with 100/zl of the sample to be tested, then incubated at 37°C for 30 min. After incubation, coverslips were washed three times with EIS and dried at 40°C. Control cover- slips were coated with EIS alone.

Aliquots (103 cells/100 tzl) of cell suspension, covering an ap- proximate area of 10 mm 2, were added to the coated coverslips. After 60 min incubation at room temperature, the coverslips were washed with EIS then timed in a 10010 formalin EIS solution and the percentage of attached ceils assessed by counting both amoe- bocytes and spherule ceils in the sample area. Cell-types were identified according to the characteristics reported by Canicatti et al [9].

Metabolic and cytoskeleton inhibitors

To test the effects of metabolic and cytoskeleton inhibitors on cell-attachment ability, a 1 mM solution of potassium cyanide, or a 1 or 5 mM Solution of sodium azide, were added to cell sus- pensions immediately before the cell adhesion tests. In addition vinblastine and cytochalasin B (Sigma), were also tested. The two latter cytoskeletal inhibitors were tested at concentrations of 2, 20 and 200/zg/ml, with a 0.1 ~/o dimethyl sulphoxide solution as the solvent. The solvent alone was used in control experiments.

Substrata

Protein measurement

To ascertain the effect of different substrata on the adhesive properties of coelomocytes, cell-free coelomic fluid was used to coat glass coverslips. Coelomic fluid was collected from coelomic cavity and centrifuged at.400 g for 10 rain at 4°C. Aliquots were dialyzed in a solution of 20 mM Tris-HCl and 500 mM NaCI (pH 7.5). A further sample of coelomic fluid was heated at 100°C for 30 min.

In addition to coelomic fluid, the coelomocyte agglutinating factor (CAF), was also used as a substrate. This material was extracted according to the method of Canicatti and Rizzo [15], then glass coverslips were coated with a 5- or 25-/~g/ml concen- tration of this preparation.

Scanning electron microscopy

Protein concentrations were determined by the method of Lowry [38] using bovine serum albumin as the standard. I I

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Coverslips with attached cells were washed in EIS then fixed over- night in 2.5°70 glut.araldehyde in 0.1 M cacodylate buffer 0.5 M NaCI solution (CABS) (pH 7.2). The coverslips were then washed three times in the buffer solution and postfixed for I h in 2070 osmium tetroxide in CABS. After washing, the ceils were de- hydrated in a graded series of acetone, critical point-dried and coated with gold. Cells were observed and photographed with a Philips 515 scanning electron microscope, operated at 20 kV.

R e s u l t s

The various numbers of each cell-type adhering to the glass coverslips after in vitro incubation clearly differed f rom one another; a smaller percentage of amoebocytes (13.3 +_ 3.4°70) attached to the glass surface than spher- ule cells (23.2 + 4.6%). The adhesion activity of each cell- type varied greatly f rom one individual to another, but amoebocytes consistently demonstrated less adhesiveness (range: 6.8 __. 1.6070 to 23.2 + 4.1070) than spherule cells (range: 14.7 ___ 2.80/0 to 34.2 _+ 1.6070). Temperature and incubation time influenced the adhesive ability of both cell- types; maximum cell-attachment was obtained at a tem- perature of 20°C and an incubation time of 60 rain.

Divalent cations enhanced cell-attachment in both cell- types (fig 1). In the presence of 5 mM calcium only a slight increase in adhesiveness could be observed (fig 1A, B), but higher concentrations (10 and 20 mM) failed to increase proportionally the number of glass-attached ceils. The ef- fect of Ca 2÷ varied with experiments and it was some- times not detectable at all. Magnesium was instead more effective in increasing the proportion of adhering cells. The maximum effect of this cation was observed at a 10-mM concentration; a higher concentration (20 mM) tended to inhibit cell attachment.

I f ceils were allowed to adhere to the glass coverslip in the presence of 5 mM EDTA, at tachment of both spher- ale cells and amoebocytes occurred normally. Increasing concentrations (10 and 20 mM) of the chelating agent produced only a slight decrease in the number of attached cells (fig 1).

Scanning electron microscopy of spontaneously attached amoebocytes revealed two distinct configurations which were respectively described as having spread and stellate forms. In figure 2a the morphology of a spread amoebo- cyte is shown. After 1 h of incubation, the whole cell as- sumed a flattened shape. The dorsal surface of the nuclear

40-

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Attaoh~ mille (%)

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Concentrations (raM)

Fig 1. Effect of Ca 2+, Mg 2+ and EDTA on coelomocyte adhe- siveness. A. Spherule cells. B. Amoebocytes. The cell suspen- sions were prepared in presence of different concentrations of both cations and chelating agents and allowed to adhere on clean glass coverslips. After 60 min the percentage of attached cells was evaluated. Mean values with SE < _+ 3.2, n = 6.

area appeared to be folded whereas the remaining area was mostly smooth. Peripherally, extensions of cytoplasmic lamellae~ sometimes arborized, were observed. Occasion- ally, many blebs of different sizes occurred on dorsal sur- faces. They were presumably granules of lysed cells which accidentally covered the cell surface.

The stellate configuration of amoebocytes attached to glass after 1 h of incubation was characterized by numer- ous filamentous extensions developed f rom the central body of the cells (fig 2b). The extending filaments were sometimes longer than 30/zm and arborized. They pro- truded both into the medium and were in contact with the substratum (fig 2b).

Adhesive spherule cells apparently did not assume a flat- tened shape. After 60 min, the cells maintained their spher-

Adhesive properties of sea cucumber coelomocytes 99

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Fig 2. Scanning electron microscopy of glass-attached sea cucumber coelomocytes, a. Spread amoebocyte. Magnification 6 100 ×. b. Stellate amoebocyte. Note the numerous filipodia (f). Magnification 2 800 x . c. Spherule cell. Magnification 7 000 ×. d. Spherule cells with microprojections (m). Magnification 5 700 ×.

ical form without signs of degranulation (fig 2c). Under these conditions, neither pseudopodia nor filipodia were seen protruding f rom these cells. Few microprojections were sometimes observed on spherule cell surfaces (fig 2d).

Spherule cell and amoebocyte attachment did not change in the presence of both cyanide (1 mM final concentra- tion) and azide (1 and 5 mM final concentrations) (table I), but was influenced by cytoskeleton inhibitors. As indicated in figure 3A vinblastine consistently reduced the number of attached cells to 20 ~zg/ml. At a similar concentration, cytochalasin B was ineffective on both spherule cells and amoebocytes (fig 3B). Inhibition of adhesion was seen only when high concentrations (200/~g/ml) of inhibitor were used (fig 3B).

The coelomocyte adhesion activity is strongly influenced by coelomic fluid preparations used as substrata. As shown in table II, in contrast to control, the percentage of at- tached spherule cells on 12/zg/100/zl coated coelomic fluid glass coverslips increased by about 50%. Amoebocytes ap-

Table I. Effect of metabolic inhibitors on coelomocyte attach- ment on glass coverslip.

Inhibitors 070 At tached cells ~ Amoebocy tes Spherule cells

None 11.8 +_ 9.5 20.0 + 9.4 KCN 1 mM 13.3 _+ 1.7 24.5 + 4.1 NaN 3 1 mM 12.3 + 1.6 19.3 + 2.7 NaN 3 5 mM 16.8 + 10.6 25.4 _+ 4.2

a Mean values __. SE, n = 6.

peared to be more sensitive than spherule cells to coel- omic fluid substratum. Their percentage of attachment on glass surface coated with 12 ~g/100/zl of CF was three- fold higher than uncoated glass coverslips (table II). The enhancer effect of CF was not influenced by extensive

100 C Canicatti et al

A: Vinblastine

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Fig 3. Effect of cytoskeletal inhibitors. Coelomocytes were al- lowed to adhere on clean glass surfaces in presence of different concentrations of vinblastine (A) and cytochalasin B (B). The data reported represent mean values with a SE < ± 4.2 for six separate experiments.

dialysis in 20 mM Tris-HCl, 500 mM NaCI (pH 7.5) nor by heating at 100°C for 30 min of the CF samples (table 11).

When purified coelomocyte agglutinating factor (CAF) was used as a substratum, an increase in adhesion was ob- served in both spherule cells and amoebocytes (fig 4). With respect to controls, an amount of 5/zg of protein per coverslip produced a consistent increase (about 50°70) in the amoebocytes ' adhesion activity, but only a small in- crease of the spherule cells' activity. Higher concentrations (25 ~g) of purified CAF did not produce further increase in amoebocyte adhesiveness, whereas in spherule cells they did (fig 4).

Table 11. Effect of coelomic fluid on coelomocyte glass-adhesion.

Substrata ° Protein content mg/ml

~o Attached cells b

Spherule cells Amoebocytes

None - 17.0 ± 4.2 18.8 ± 3.1 CF N 0.12 40.7 ± 1.9 60.1 ± 4.0 CFTRtS 0.12 37.9 ± 2.1 59.4 ± 4.5 CF~00. c 0.12 30.4 ± 2.0 60.0 ± 3.2

a C F N - non treated coelomic fluid; C F T R I S = 20 mM Tris- HC1, 500 mM NaC1 (pH 7.5) dialyzed coelomic fluid; CFl00. c = coelomic fluid heated at 100°C for 30 rain. b Mean values ± SE, n = 6.

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Fig 4. Effect of purified CAF on adhesive activity of coelomo- cytes. The columns in the histogram represent the mean values wi thaSE < _+ 2.8, n = 6.

D i s c u s s i o n

H o l o t h u r i a p o l i i amoebocytes and spherule cells attached and spread in vitro on glass coverslips. As for other in- vertebrate blood ceils [7, 16, 17], the sea cucumber coe- lomocytes require divalent cations tO bind to the substratum. Magnesium was found to be more effective than calcium in facilitating adhesion of both amoebocytes and spherule cells to the glass surface. These results ap- peared to contradict the general recognition of the specif- ic importance of Ca 2+ in cell adhesion [18] although Mg2+-dependent cell a t tachment was demonstrated for fibroblasts f rom a pr imary culture of chick embryonic sclera [19].

From the SEM observations it is evident that two dis- tinct arnoebocytes bind to glass surfaces. The first type spread and assumed a flattened configuration, while the second type extended numerous protrusions both into the medium and in contact with the glass surface assuming a steUate configuration. The flattened amoebocyte morpho- logy is shared by almost all the spreading cells studied so far [17, 18, 20] and most probably constitutes a stable stage

Adhesive properties of sea cucumber coelomocytes 101

of a process including initial attachment, spreading and organization of cytoskeletal fibers. The stellate morpho- logy, however, appears to be a characteristic of the sea cucumber amoebocytes. It results from glass-attachment of the f'dipodial stage of these cells. Echinoderm amoebocytes occur in two distinguishable stages, petaloid and filipodi- al [21, 22]. Transformation from one type to the other oc- curs physiologically and is induced by different stimuli [14]. The generally fast process consists of a complete transformation of the characteristic petals of petaloid amoebocytes into filipodia that can reach a length of 40/zm [23, 24]. The different attachment behaviours of amoebocytes observed here could implicate a diverse physiological engagement of these cells in the H polii defence repertoire. Flattening amoebocytes could be responsible for nodule formation during encapsulation, since layers of elongated and extremely flattened amoe- bocytes constitute the reactive nodular cells surrounding foreign material [5]. On the other hand, the stellate con- figuration could be implicated in clot formation, since an intricate network of filipodial protrusions of amoebocytes has been related to clotting reaction [11]. The other Hpoli i cellular category exhibiting adhesive properties was represented by spherule cells. In contrast to amoebocytes, however, they were never seen to spread.

As for crayfish haemocytes [7], the binding of coelomo- cytes to glass seems not to require any nietabolic energy. Nevertheless, it was influenced by the anti-tubulin cytoskeleton inhibitor, vinblastine. Cytochalasin B, which inhibits cytokinesis by reversible disruption of contractile microfilarnents [25], did not influence cellular attachment, suggesting that, as for flbroblastic cells [26-28], microfila- ments are not essential for initial contact with substratum.

Ceils adhere to different substrata via adhesive molecules [29]. For most of them, fibronectin [18, 30, 31], laminin [32, 33] and selectin [34], the central function in adhesion is well recognized. In H polii molecules able to mediate coelomocyte adhesion were recognized in a series of ex- periments with coelomic fluid and purified coelomocyte agglutinating factor. The latter factor is a 220-kDa pro- tein, released in vitro by coelomocytes [13] which ag- glutinate the sea cucumber cells, and is thus thought to be involved in the first phase of clotting events [15]. In vivo, the molecule is most likely released into coelomic fluid thus explaining the enlaancing of cell adhesion ac- tivity produced by coelomic plasma. CAF promotes cell adhesion in a dose-dependent fashion but its nature re- mains unclear. It could represent a fibronectin-like molecule, since a protein with a similar molecular mass (220 kDa) and able to mediate in vitro adhesion and spreading of baby hamster kidney cells was isolated from the ovary of sea urchin Pseudocentrotus depressus [35, 36]. In other invertebrates, an aggregating factor guides adhe- sive interactions between cells [37]. In Limulus, attach- ment, migration and spreading of amoebocytes to glass are most probably regulated by factors contained in the cells [16]. Coagulogen was proposed as a possible candi- date [17]. The same is not true for crayfish blood cells in which a non-coagulogen-like 76-kDa cell adhesion factor is responsible for attaching and spreading of haemocytes [22].

Work is now under way to explore both possibilities.

Acknowledgments

This work was supported by MURST grants. We are grateful to Mr M Moretti for his technical assistance.

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