ISJ106.PDF


 

ISJ 2: 124-131, 2005               ISSN 1824-307X 
 
 

Research Report 
 
 

Wound repair in the marine worm Sipunculus nudus (Sipunculidae) 
 
 
G D’Ancona Lunetta 
 
Institute of Histology and Embryology University of Palermo, Italy 
 
 

Accepted September 14, 2005 

 
Abstract 
The cells and molecules involved in the wound healing of Sipunculus nudus were studied. An incision, 
5 mm in length, was cut longitudinally at a site opposite the anus and 10 mm from the introvert. The 
histological study performed at different times showed an involvement of both Type I and Type II 
granulocytes in the process of healing. The former were capable of extracellular digestion and they 
were immunoreactive to anti-IL-4, -IL-10 and -epidermal growth factor (EGF) antibodies (Abs); the 
latter were involved in the synthesis of connective tissue from 24 h after the incision, thereby causing 
the initial closing of the wound. After 70 h, a continuous layer of Type II granulocytes was found on the 
sides of the wound where the future muscle tissue would be formed; many of these granulocytes had 
been partially degranulated. It was not possible to establish any existing relationship between the 
functions of Type I granulocytes and their reactivity to anti-IL-4, -IL-10 and  -EGF Abs. 
 
Kew words: marine worm; Sipunculus nudus; wound repair; cytokines 

 
 
 
Introduction 
 
The process of wound healing has been the subject of 
intensive research, mainly in vertebrates (Redd et al., 
2004; Harvey, 2005; Whitney, 2005). With regard to 
invertebrates, Kindred (1924) observed that the 
removal of a fragment of the body wall in echinoderms 
led to the healing of the wound, by the proliferation 
and infiltration of cells from the surrounding 
connective tissue. In Asterias the aggregation of 
coelomocyte and of amoebocytes from adjacent 
tissue was seen to contribute to the wound healing 
(Anderson, 1962, 1965). In the sea cucumber 
Stichopus tremulus numerous morula cells, found in 
proximity of the incision, were supposed to play a 
significant role in healing wounds, and to be 
homologous with the vertebrate mastocytes 
(Rollefsen, 1965). In contrast, in Stichopus 
badionotus, after superficial cutaneous incisions 
Cowden (1968) observed a rapid repair with complete 
fibrogenesis without the intervention of morula cells.  
 
 
Corresponding Author: 
D’Ancona Lunetta G 
Istituto di Istologia ed Embriologia, Dipartimento di 
Biologia, Università di Palermo, 
viale delle Scienze, 90123 Palermo, Italy 
E-mail: dancona@unipa.it 

The migration of epidermal and pigment cells from the 
periphery of the wound margin in Thyone briareus 
caused re-epithelialization in the absence of evident 
mitotic activity, an morula cells seemed to be involved 
in this migration although their precise role is unknown 
(Menton and Eisen, 1974).  Fibroblast-like cells, 
pigment cells and numerous coelomocytes were the 
first cells to arrive at wound sites in the Holothuria polii 
and the newly formed collagen fibres were 
synthesized by Type II spherula cells.  Cutaneous 
lesions performed after antigenic stimulation healed 
more slowly than controls since Type II spherula cells 
were also actively involved in the immune reaction by 
forming brown bodies in cooperation with the 
amoebocytes (D’Ancona, unpublished). Consequently, 
in this case, a lower number of Type II spherula cells 
are available for the synthesis of connective 
components (Canicattì and D’Ancona, 1989).    

Recently, Franchini and Ottaviani (2000) have 
studied the effects of platelet-derived growth factor 
(PDGF-AB) and transforming growth factor (TGF-β)1 
in the repair mechanism of wounds in the mollusc 
Limax maximus. The main repair stages include an 
initial infiltration phase in which the hemocytes migrate 
and stratify at wound margins, actively phagocitize cell 
debris and damaged tissue and were immunoreactive 
to anti-IL-1α, -IL-8 and -tumor necrosis factor (TNF)-α 
antibodies (Abs). This was followed by the formation of 
granulation tissue, the synthesis and depositing of 

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extracellular matrix components, such as fibronectin, 
collagen fibres and reticular fibres.  Finally, re-
epithelialization of the wound occurred. The 
exogenous administration of PDGF-AB and TGF-β1 
stimulated the tissue healing process though a 
general acceleration of the activities involved.   

In the coelomic fluid of S. nudus two types of 
granulocytes (Type I and Type II), are distinguishable 
on the basis of their morphology and chemical 
composition. Type I granulocytes do not have a 
phagocytic capability, even though they contain lytic 
enzymes; Type II granulocytes show phagocytic 
activity. Furthermore, haemerythrocytes, signet-ring 
cells, urna cells complexes, empty vesicles, vesicle 
fragments, laminar structures, aggregates of stem 
cells and brown bodies were also found (D’Ancona et 
al., 2004). In the present paper the mechanism of 
wound repair in Sipunculus nudus was studied. 
Furthermore, it was also examined if cells reactive to 
anti-IL-4, -IL-10 and -EGF Abs were present and 
involved in the wound healing of the marine worm.  
 
 
Materials and Methods 
 

  Twenty adult specimens of Sipunculus nudus 
were incised with a scalpel in the frontal part of the 
body, producing a wound in a site opposite to the 
anus and 10 mm from the introvert. The incision, 5 
mm in length, was performed longitudinally and it 
involved the integument and the circular muscle 
structure below. The animals were sacrificed and fixed 
in Bouin mixture at different times after the incision (3, 
15, 18, 22, 24, 71 and 96 h) and unwounded 
specimens were used as controls. Fragments 
including the wound and 2 mm of surrounding healthy 
tissue were included in paraffin and 7 µm thick 
sections were stained with Gomori triple staining (the 
connective tissue resulted green and the Type I 
granulocytes red) and Alcian blue-PAS (Type II 
granulocytes stained green and pink) (Ganter and 
Jòlles, 1969; Mazzi, 1977). 
   The immunocytochemical reactions were performed 
by incubating sections for 1 h at room temperature 
(RT) in mouse primary monoclonal Abs (Euroclon, 
Celbio, Italy), raised against IL-4, IL-10 and EGF, 
diluted 1:100 in phosphate-buffered saline (PBS) 
(1.37 M NaCl, 0.03 M KCl, 0.015 M  KH2PO4 and 
0.065 M Na2HPO4), rapidly washed in PBS, incubated 
for 30 min at RT in biotinylated goat anti-mouse 
immunoglobulins (Kit DAKO cytomation, Denmark), 
washed in PBS, incubated in streptavidin peroxidase 
conjugate for 30 min at RT. After washing, sections 
were stained for 15 min in the chromogen 
aminoethylcarbamate. Sections incubated with normal 
non-immune rabbit serum were used as controls. 
 
 
Results  
 

In physiological conditions the body walls of S. 
nudus consist of a cubic epithelium, secreting an outer 
cuticle, a derma, a layer of circular muscle, a layer of 
longitudinal muscle and the peritoneum. The derma 
contains fine fibres, connective cells and coelomatic, 
longitudinal canals, which communicate with each 
other and with the general coelom (Hyman, 1959).  

As far as the wounded specimens are concerned, 
3 h after the incision, the wound did not show any 
signs of healing, the layers of muscle and connective 
tissue were still damaged and the cuticle was missing. 
On the outer part of the wound, coelomocytes were 
present, most of which were acidophilic Type I 
granulocytes (Fig. 1). Furthermore, Type II 
granulocytes and a few haemerythrocytes were 
observed. Three h after the incision, an intense 
immunoreaction with anti-EGF Ab was observed in 
Type I granulocytes, including those which were 
present in the coelomatic cavity (Fig. 2).  Type II 
granulocytes and haemerythrocytes were negative. No 
changes in the response were observed up to 15 h 
after the incision, but for an increase in exocytised 
acidophilic material.  

Eighteen h after incision, new muscle fibres were 
not observed but fine connective fibres were evident. 
Both partially degranulated Type I granulocytes and 
Type II granulocytes were present, in addition to 
transparent spherical cells on the outer part and the 
sides of the wound.  

Twenty-two h after incision, the wound had been 
externally closed up by fine collagen fibres and 
internally by numerous coelomocytes 
(haemerythrocytes, Type I granulocytes and various 
Type II granulocytes). At this time, EGF-like material 
was found in all Type I granulocytes, while other 
coelomocytes were negative (Fig. 3). 

Twenty-four h after incision, the wound had 
closed: on the internal part of the wound, acidophilic 
Type I granulocytes were present and the circular, 
muscle tissue was interrupted towards the central part 
of the wound.  In its place there was newly 
synthesized connective tissue with numerous Type I 
and Type II granulocytes nearby. Type I granulocytes 
were highly reactive to the anti-IL-4 Ab, while Type II 
granulocytes, were less numerous and negative (Figs 
4, 5).  

In incisions of the entire animal wall, both the 
derma and muscle tissue were interrupted and the 
circular muscle fibres displayed rounded extremities, 
which were covered by connective tissue (Fig. 6). The 
wound had been closed by various thin, connective 
lamina distributed over 2-3 layers at the extremities. 
Each lamina contained Type II granulocytes and a few 
transparent cells (Fig. 7). Some Type II granulocytes 
start to loose their basophilic core, while others devoid 
of the basophilic granules, were amoeboid in shape 
with a flattened nucleus and were involved in collagen 
fibre production (Fig. 8). Twenty-four h after the 
incision, large spaces containing degranulating Type I 
granulocytes were highlighted near the tissue on the 
internal part of the wound, and these degranulated 
cells flattened and formed a sort of barrier that trapped 
haemerythrocytes and Type II granulocytes  (Fig. 9). 
Type I granulocytes located towards the coelomatic 
cavity were almost degranulated with a weak reaction 
to anti-IL-10 Ab.  Type II granulocytes did not react 
with this Ab, while degranulated material resulted 
highly immunopositive (Fig. 10). Ninety-six h after the 
incision, the walls of S. nudus had been reconstituted 
but the connective tissue was not always well 
compacted and the muscle fibres did not show their 
typical circular arrangement (Fig. 11).  In the lateral 
parts of the wound, the muscle fibres were interrupted 
and a great number of “spongy” Type II granulocytes,  

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Fig. 1 Histological section 3 h after incision, stained with Gomori triple staining. Note, on the outer part of the 
wound (op), coelomocytes (←) with acidophilic material and degranulated acidophilic material (*). The connective 
tissue and muscle tissue (**) are still damaged and the cuticle (c) is also missing. Coelomatic cavity (cc). 
 
 
 
 

 
 
Fig. 2 Immunocytochemical reaction with anti-EGF Ab 3h after incision. Positive Type I granulocytes (g1); 
negative Type II granulocytes (g2) and haemerithrocytes (h) in the coelomatic cavity. 

 
 
 
 
 

 
 
Fig. 3 Immunocytochemical reaction with anti-EGF Ab 22 h after incision. Positive Type I granulocytes (g1) and 
degranulated material (dm); negative haemerithrocytes (h). Outer part of the wound (op), Cuticle (c). 

 

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Fig. 4 Immunocytochemical reaction with anti-IL-4 Ab 24 h after incision. Positive Type I granulocytes (g1) are 
present in the connetive tissue (ct). Coelomatic cavity (cc); longitudinal muscle (lm), outer part of the wound (op). 

 
 

 
 

Fig. 5 Detail of Fig. 4. Type I granulocytes (g1) positive and Type II granulocytes (g2) negative to anti-IL-4 Ab.  
Collagen fibres (cf). 
 
 

 
 
 
Fig. 6 Histological section 24 h after wound, stained with Gomori triple staining. The incision concerned the entire 
thickness of the animal wall. The wound is closed by connective lamina (cl). Longitudinal canal (lc); coelomocytes 
and acidophilic material (c); circular muscle fibres (cm); coelomatic cavity (cc); longitudinal muscle (lm). 

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Fig. 7 Detail of Fig. 6. Note longitudinal canal (lc); degranulated acidophilic material (*); coelomocytes (c); 
connective lamina distributed over 2-3 layers (cl); circular muscle fibres with rounded extremities (cm); coelomatic 
cavity (cc); longitudinal muscle (lm); outer part of the wound (op). 
 
 

 
 
 Fig. 8 Detail of Fig. 6. External surface of the wound. Note type I granulocyte (g1); connective tissue (ct) where 
Type II granulocytes (g2a) displayed their basophilic core and Type II granulocytes shaped like a triangle (g2) are 
producing thin bundles of collagen fibrils (cf).    

 
 

 
 
 Fig. 9 Histological section stained with Gomori triple staining 24 h after a deep incision. Note a barrier made up 
of flattened Type I granulocytes (g1), which have trapped haemerythrocytes (h), type II granulocytes (g2) and 
degranulated material (dm). 

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Fig. 10 Immunocytochemical reaction of section with anti-IL-10 Ab 24h after incision. Partially degranulated Type 
I granulocytes (g1) were moderately reactive whilst the degranulated material (dm) was highly reactive. Negative 
Type II granulocyte (g2). Note Type I flattened granulocytes (fg1); connective tissue (ct); outer part of the wound 
(op); coelomatic cavity (cc). 
 

 

 
 
 
Fig. 11 Histological section 96 h after incision stained with Gomori triple staining. The wound is partially healed. 
Note cuticle (c); granulocytes; poorly compacted collagen fibrils (cf); circular muscle fibres (cmf) being formed; 
coelomic cavity (cc); longitudinal muscle (lm). 
 
 

 
 

Fig. 12 Detail of Fig. 11 in the lateral parts of wound. Note the interrupted muscle fibres (mf) and a great number 
of spongy type II granulocytes (g2), separated by connective fibres (cf). 
 
 

 
 

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separated by connective material, formed a base onto 
which the muscle fibres were formed (Fig. 12).  
Elongated Type I granulocytes, with varying amounts 
of acidophilic granules, were found between the 
forming muscle fibres.    
 
 
Discussion 
 

The histological study performed 3-96 h after the 
incision in S. nudus revealed that Type I and Type II 
granulocytes from the coelomatic cavity are 
responsible of the wound repair. Type I granulocytes 
are the first cells to be activated and they 
degranulated thereby causing the transfer of their 
material to the surrounding area and, in particular, on 
Type II granulocytes. The contribution of the  
coelomocytes was not always the same. In most 
cases, the coelomocytes migrated from the coelomic 
cavity to the lesion zone, due to the probable 
presence of various chemotactic substances 
produced in situ (Abercrombie, 1972; Postletwaite, 
1976). Type I granulocytes contain lysosomal 
enzymes  (Matozzo et al., 2001; D’Ancona et al., 
2004) that could act in the digestion of cell debris and 
damaged tissue. The urna cell complexes present in 
the coelomatic liquid of S. nudus (D’Ancona et al., 
2004) and endowed with phagocytic activity (Bang 
and Bang, 1962) did not participate in wound repair 
process. Unlike the hemocytes found in the wound 
repair of L. maximus (Franchini and Ottaviani, 2000), 
Type I granulocytes do not take part directly by means 
of phagocytosis but they secrete enzymes into the 
extracellular environment. 

Type I granulocytes also have an important 
hemostatic function. Indeed, they move from the 
coelomatic cavity towards the wound in great amounts 
in order to create an aggregate that can prevent the 
passage of coelomatic material towards the outer and 
of pathogens towards the inner side of the body.  
These cells flattened after degranulation, stuck along 
the lower margins of the wound and form a lamina 
which entrap other cells, especially haemerythrocytes.  
Many of these, in turn, flocked around the wound with  
the aim of supplying oxygen, which is necessary for 
metabolic activity (Steins et al., 2001).   

  Rollefsen (1965) hypothesized that morula cells, 
present in the hydrovascular system and dermal 
connective tissue of S. tremulus, can be involved in 
the production of the intercellular substance of 
connective tissue, as they are both metachromatic 
and PAS positive. Morula cells may form fibres 
(Endean, 1966) which are responsible for connective 
tissue growth and repair (Smith, 1981). In other 
echinoderms polysaccharides and proteins are 
present in the granular inclusions of spherula cells 
(Endean, 1958; Hetzel, 1963; Johnson, 1969; 
D’Ancona and Canicattì, 1990). Type II  granulocytes 
in S. nudus showed the same cytological 
characteristics of the morula cells of S. tremulus and 
H. poli Type I spherula cells (D’Ancona and Canicattì, 
1990). They also have the same histochemical affinity 
of connective tissue and it can, therefore, be 
hypothesized that Type II granulocytes may be the 
main cell producers of connective material.   

The granulation tissue, typical of wound healing 
in vertebrates, was also found in L. maximus 

(Franchini and Ottaviani, 2000). In this mollusc the 
main cell type involved in the different phases of repair 
process is the hemocyte. Indeed, this cell was 
immunoreactive to cytokines, was able to phagocytize 
cell debris and damaged tissue and showed fibroblast-
like activity, as found   by Sminia et al. (1973) in L. 
stagnalis. 

In S. nudus numerous Type I granulocytes are 
found everywhere and they continuously exocytise 
acidophilic material between other cells and  muscle 
fibres. Moreover these cells contain hydrolytic 
enzymes, able to remove cell debris and damaged 
tissue, and were also immunoreactive to EGF-, IL4- 
and IL10 Abs. In marine worm it can be supposed, as 
in vertebrates, that in the first hours after incision, an 
inflammatory response occurs, involving cellular 
proliferation of granulocytes (D’Ancona et al., 2004).   

In mammals and some invertebrates, growth 
factors such as PDGF and TGF-β, play an important 
role in healing wounds as they are chemotactic and 
proliferative factors (Seppa et al., 1982; Deuel et al., 
1982; Senior et al., 1983; Postlethwaite et al., 1987; 
Wahl et al., 1987; Clark et al., 1997; Ottaviani et al., 
1997; Franchini and Ottaviani, 2000).  The presence of 
EGF-, IL-4- and IL-10-like material in S. nudus does 
not exclude the possibility that other cytokines or other 
factors, found in other invertebrates, could also be 
present in Type I granulocytes, and that they could be 
directly involved in the process of wound healing.  
However, it is so far impossible to attribute a specific 
function to EGF-, IL-4- and IL-10-like material in the 
process of the healing of wounds in S. nudus. 

 
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