AMUR.

ZOOU, 23:145-156 (1983)

Inflammatory Responses in Annelids1

ELIZABETH A. STEIN AND EDWIN L. COOPER
Department of Anatomy, School of Medicine, University of California
Los'Angeles, California 90024

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SYNOPSIS. The cellular inflammatory responses of annelids, which include chemotaxis,
phagocytosis, encapsulation, and various phases of wound healing, are effected by coe-
lomocytes, particularly the hyaline amoebocytes. Coelomic epithelial cells and specialized
nephridial cells also participate in phagocytosis. A number of factors related to inflam-
mation in vertebrates have been identified in annelids, i.e., histamine, kallikrein, lysosomal
enzymes, agglutinins, lysins and antibacterial substances. However, their precise function
in annelid inflammatory responses has yet to be determined.

INTRODUCTION
Earliest accounts of inflammatory reac-
tions in annelids were those of Metchnikoff
(1884, 1893) and were followed soon
thereafter by studies from a number of
other investigators (Kowalevsky, 1889;
Keng, 1895; Cuenot, 1898; Caullery and
Mesnil, 1899; Willem and Minne, 1899)
who observed that wandering cells of the
coelomic cavity, coelomocytes, were
responsible for removal or inactivation of
infectious organisms such as bacteria, para-
sitic protozoa and nematodes. Cameron
(1932) subsequently described in detail the
responses of earthworms to a variety of
inflammatory agents and recorded the
series of stages involved in wound healing
and tissue repair. More recently, investi-
gations into immune responses of earth-
worms and polychaetes have extended our
understanding of various elements of the
inflammatory process in annelids (for
reviews see Cooper, 1976; Dales, 1978;
Cooper and Stein, 1981; Dales and Dixon,
1981; Valembois, et ai, 1982).
In vertebrates, the dynamics of inflam-
mation have been extensively investigated
and characterized by specific morpholog-
ical and physiological events (Ryan and
Majno, 1977). In invertebrates, such events
have not been as well denned. However,
certain aspects of cellular responses—
chemotaxis, phagocytosis, encapsulation
and wound healing—have been investi-
1
From the Symposium on Comparative Aspects of
Inflammation presented at the Annual Meeting of the
American Society of Zoologists, 27-30 December
1981, at Dallas, Texas.
gated in varying degrees. Humoral
responses involving the intricate network
of myriad soluble factors (vaso-active sub-
stances, chemotactic factors, complement
components, etc.) that operate in verte-
brate inflammatory reactions remain vir-
tually uninvestigated in invertebrates, and
it is not yet clear to what degree such fac-
tors operate in annelid responses.
The annelids consist of three classes:
Polychaeta, Oligochaeta, and Hirudinea.
The polychaetes are primarily marine
worms, the oligochaetes are both aquatic
and terrestrial and include the familiar
earthworms, while hirudineans or leeches
are primarily parasites. We will consider
each of the three groups when discussing
different components of annelid inflam-
matory reactions, and will compare their
responses whenever possible. Since earth-
worms have been the most widely studied
they will be discussed most frequently, but
polychaetes have also received consider-
able attention. Information on leeches, in
most instances, has been sparse, but will be
included whenever possible.
CELLULAR RESPONSES TO INFLAMMATION
Annelid leukocytes
The coelomocytes of annelids vary widely
in morphology, not only between classes
but also between species of the same fam-
ily. Despite this diversity of structure, two
major categories are present in all three
annelid groups, amoebocytes and eleo-
cytes.
(1) Amoebocytes are generally motile,
phagocytic cells that exhibit varying
amounts of granulation. In earthworms, the
145
146 E. A. STEIN AND E. L. COOPER

relatively non-granular forms, or hyaline case of eleocytes, not directly involved in

amoebocytes, range in size from small cells inflammatory processes (Fange, 1950).
of 5-8 Mm diameter, with a small cyto- Polychaetes also possess both hyaline and
plasmic volume and high nucleocyto- granular amoebocytes; as in the case of oli-
plasmic ratio, to larger cells (up to 30 urn gochaetes, their relative proportions in the

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in diameter) with greater cytoplasmic vol- coelomic fluid, as well as their morphology,
umes. Although classified as non-granular, vary widely among different species (Dales
hyaline amoebocytes usually do contain and Dixon, 1981). Although some poly-
varying numbers of small granules. In some chaete amoebocytes are spherical or ovoid,
instances, these granules are identifiable as they are more often fusiform or spindle-
lysosomes, staining cytochemically for the shaped. The cytoplasm characteristically
enzymes acid phosphatase and /3-glucuron- contains prominent bundles of microfil-
idase (Valembois, 1971; Linthicum et al., aments (Baskin, 1974; Dales and Dixon,
1977; Stein and Cooper, 1978). 1981). Granular amoebocytes contain dis-
In Lumbricus terrestris, one of the most tinctive membrane-bound, electron-dense
thoroughly studied of the annelid worms, granules which Baskin (1974) suggested
two types of hyaline amoebocytes have been might be lysosomes, but whose function
distinguished, based on differing morpho- remains obscure.
logical and physiological properties. These In leeches, amoebocytes are relatively
have been referred to as basophils (also as small (6-10 fim diameter) in comparison to
lymphocytic coelomocytes, by ultrastruc- those of other annelids. Although their
tural critera) and neutrophils (or type I coelomocytes have been studied much less
granulocytes, in reference to the abund- extensively than those of the other two
ance of small cytoplasmic lysosome-like groups, hirudinean amoebocytes appear to
granules observable by electron micros- be more structurally homogeneous than
copy) (Linthicum et al, 1977; Stein et al., those of earthworms or polychaetes (Saw-
1977). Basophils are more numerous than yer and Fitzgerald, 1981).
neutrophils, occurring in the coelomic fluid (2) Eleocytes (chloragogen cells) are pres-
with a frequency of approximately 65%, ent in nearly all annelids (Ratcliffe and
while neutrophils are approximately 18%. Rowley, 1981). They are the dominant
Granular amoebocytes are also found con- coelomocyte type in some species but in
sistently within the coelomic fluid of most others occur in lower proportions (<2% in
annelids. Size and numbers of granules Lumbricus terrestris, approximately 10% in
vary, and some species contain at least two Eiseniafoetida). Characteristically, they are
subpopulations of granular amoebocytes. large cells (up to 40-60 fim diameter) con-
Granule staining properties are also vari- taining vesicles or granules (chlorago-
able (although acidophilic granules have somes) filled with lipids or lipid-like mate-
been the most frequently reported) and in rial and protein. Glycogen is usually
most earthworms the function of the gran- abundant but the number and content of
ular material is unknown (Cooper and the cytoplasmic inclusions may vary with
Stein, 1981; Dales and Dixon, 1981). Mem- the nutritional state of the animal (Valem-
bers of some oligochaete families, partic- bois and Cazaux, 1970; Stein and Cooper,
ularly the Megascolecidae and Enchytrae- 1978; Dales and Dixon, 1981). Eleocytes
idae, also possess other cell types which do of polychaetes frequently contain large
not fit the general categories given above. vacuoles, and the cells undergo both mor-
From these families, coelomocytes referred phologic and metabolic changes (particu-
to as "linocytes" and "lamprocytes" (the larly related to protein synthesis) that
latter also called "mucocytes") (Hess, 1970) accompany different stages in the animal's
are the dominant cell types in some species life cycle. The eleocytes of leeches, as with
(Goodrich, 1897; Ohuye, 1937; Fange, their amoebocytes, tend to be somewhat
1950). Such cells have generally been con- smaller than those of earthworms or poly-
sidered nutritive in function and, as in the chaetes (Sawyer and Fitzgerald, 1981).
 ANNELID INFLAMMATORY RESPONSES 147

TABLE 1. Cell types participating in annelid inflammatory responses.

Annelid class
Response Polychaeta Oligochaeta References

Chemotaxis Not studied Primarily neutro- Not studied Marks et ai, 1979
phils (hyaline

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amoebocyte) in L.
terrestris
Phagocytosis Amoebocytes, eleo- Primarily amoebo- Primarily amoebo- For reviews see:
cytes and fixed cytes and fixed cytes and fixed Dales and Dixon,
phagocytes phagocytes; hya- phagocytes; eleo- 1981; Cooper and
line amoebocytes cytes may ingest Stein, 1981; Saw-
more active than small particles yer and Fitzgerald,
granular; eleocytes 1981
rarely active
Encapsulation Coelomocytes (types All coelomocyte Not studied For reviews see:
not specified) types Dales and Dixon,
1981; Cooper and
Stein, 1981
Wound closure All coelomocyte All coelomocyte Amoebocytes; mus- For reviews see:
types; muscle con- types; muscle con- cle contractions Dales and Dixon,
traction; later epi- traction; later epi- 1981; Cooper and
thelial covering thelial covering Stein, 1981; Saw-
yer and Fitzgerald,
1981
Wound repair Amoebocytes; possi- Primarily amoebo- Amoebocytes For reviews see:
(removal of bly eleocytes cytes Dales and Dixon,
dead and frag- 1981; Cooper and
mented cells) Stein, 1981; Saw-
yer and Fitzgerald,
1981

Although eleocytes have been proposed to
be primarily nutritive in function (Lieb-
mann, 1942; Valembois, 1971a), in poly-
chaetes they may also be phagocytic, and
participate in disposing of disintegrating
muscle fibers that result from develop-
mental changes during metamorphosis
(Baskin, 1974).
In addition to the previously described
coelomocyte types, some polychaete species
possess hemoglobin-containing cells, or
erythrocytes. In most species, erythrocytes
are specialized for transport or storage of
oxygen and are not believed to be directly
involved in immune or inflammatory
responses (Hoffman and Mangum, 1970).
Chemotaxis
The initial cellular reaction to injury or
infection is an infiltration of leukocytes into
the affected area (Ryan and Majno, 1977).
In vertebrates, the signal for this response
is generated by the release of soluble chem-
ical factors from injured tissue or, in the
case of infection, by substances released
during the interaction of pathogens with
leukocytes or serum components (e.g.,
complement). The release of such soluble
agents results in the directed migration of
leukocytes—chemotaxis—along the con-
centration gradient toward the source of
the stimulus. Chemotaxis has been inves-
tigated in only one annelid species, Lum-
bricus terrestris (Table 1) (Marks etal, 1979).
The coelomocytes of Lurnbricus were found
to migrate in vitro toward bacteria and for-
eign tissue that consisted of body wall
explants from the earthworms Eisenia foe-
tida and Pheretima sp., and an insect, Tene-
brio molitor. The magnitude of response to
bacteria was in direct proportion to bac-
terial concentration. Chemotaxis toward
body wall tissues varied with the species of
the tissue donor and was in inverse pro-
portion to phylogenetic relatedness. The
chemotactant from Eisenia was considered
148 E. A. STEIN AND E. L. COOPER

and Stein, 1981; Dales and Dixon, 1981;

Sawyer and Fitzgerald, 1981). In earth-
worms, the coelomic cavity communicates
with the external environment via dorsal
pores and nephridiopores, so that the coe-
lomic fluid often contains small numbers
of bacteria, fungal spores, protozoa and

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nematodes (Keng, 1895; Cuenot, 1898;
Cameron, 1932; Marks and Cooper, 1977).
Detailed investigations by Cameron (1932)
with lumbricid worms established the rel-
ative phagocytic potentials of different coe-
lomocyte types. He found that hyaline
amoebocytes were the most actively phago-
cytic, granular amoebocytes less so, and
eleocytes inactive. India ink, carmine and
colloidal iron were rapidly phagocytosed
while some particles, such as oil and fat
FIG. 1. Neutrophil of Lumbricus terrestris containing droplets, were rarely ingested. Recogni-
phagocytosed yeast (arrows). N, nucleus. Wright's tion specificity was also exhibited in the
stain. X2,500. (Reproduced with permission from Stein phagocytosis of sperm from various animal
and Cooper, 1981.)
species, since homologous sperm were less
frequently phagocytosed than those from
to be a low molecular weight protein. Lum- other species.
bricus body wall tissue appeared to contain In Lumbricus terrestris, both types of hya-
an inhibitory factor. Neutrophils were the line amoebocytes are highly phagocytic
predominant responding coelomocyte type (Fig. 1) while granular amoebocytes (acid-
and constituted 92-94% of the migrating ophils and granulocytes) in accordance with
cells. Cameron's observations, are less phago-
cytic but still moderately active (Stein et al.,
Phagocytosis 1977). Opsonins in the coelomic fluid of
Phagocytosis, the most fundamental and Lumbricus facilitate phagocytosis of yeast
ubiquitous component of immune and by neutrophils but not by basophils or
inflammatory responses, occurs in all anne- granular amoebocytes (Stein and Cooper,
lid species that have been studied and is 1981). In this instance, the overall phago-
effected by a number of different cell types. cytic capacity of Lumbricus is not greatly
Amoebocytic coelomocytes have been the affected by coelomic fluid factors, since
most widely studied and are perhaps the neutrophils constitute less than 20% of the
most important in terms of defense poten- coelomocyte population (Stein et al., 1977).
tial, by virtue of their ability to infiltrate The coelomocytes of polychaetes are
all tissues and reach all parts of the body. actively phagocytic and have been observed
In addition to coelomocytes, cells of the to ingest a wide variety of particles (for
coelomic epithelium may be phagocytic review, see Dales and Dixon, 1981). Amoe-
(Metchnikoff, 1893; Cuenot, 1898; Cam- bocytes of Arenicola marina phagocytose
eron, 1932) as well as certain types of mammalian erythrocytes and bacteria both
nephridial cells. In leeches, botryoidal and in vivo and in vitro, and stain strongly for
vaso-fibrous tissue of the coelomic sinuses the lysosomal enzyme acid phosphatase
are also phagocytic and have been com- (Dales and Dixon, 1981). Amoebocytes also
pared to the reticuloendothelial system of clear the coelom of redundant gametes
vertebrates (Bradbury, 1959). after the spawning season by phagocytos-
Phagocytosis by coelomocytes has been ing effete oocytes and sperm (Siedlecki,
studied extensively in many annelid species 1903; Dehorne, 1922; Dales, 1950). Unlike
(for reviews see Stephenson, 1930; Cooper earthworms, the eleocytes of polychaetes
 ANNELID INFLAMMATORY RESPONSES 149

may also be phagocytic, and participate in lomic lining toward the thread and occa-
removal of disintegrating muscle cells from sionally containing small blood vessels.
the coelom during metamorphosis (Baskin, Frequently, such encapsulation results in
1974). the formation of permanent structures in
Although amoebocytes of leeches are the coelom called "brown bodies" that tend
known to be phagocytic, few definitive to accumulate in the terminal segments of

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studies have been performed. They ingest the worm.
bacteria as well as inert particles such as The ultrastructure of brown body cap-
carmine and India ink (Willem and Minne, sules surrounding nematodes was described
1899). As in the case of polychaetes, eleo- by Poinar and Hess (1977) as consisting of
cytes may also be moderately phagocytic two layers: a non-cellular inner layer
(Willem and Minne, 1889). believed to be derived from the coelomic
fluid, and an outer, cellular layer. The cel-
Encapsulation lular layer contained at least four coelo-
Objects that are too large to be phago- mocyte types, consisting of both hyaline
cytosed, such as protozoa or parasitic and granular cells. Light-microscopic stud-
worms, are usually surrounded by large ies have indicated that brown bodies fre-
numbers of coelomocytes (primarily amoe- quently contain eleocytes and chlorago-
bocytes) and are thus encapsulated. In oli- somes (Duprat and Bouc-LaSalle, 1967;
gochaetes, nematodes that frequently Semal van-Gansen, 1956).
invade the coelomic cavity are encapsu- Encapsulation of parasites within poly-
lated and subsequently die within the cap- chaetes varies with the type of parasite and
sule (Metchnikoff, 1893; Cameron, 1932). species of worm (Dales and Dixon, 1981).
Metchnikoff (1893) reported that adult Encapsulation of parasitic trematodes has
gregarines were encapsulated and killed by been observed in Poecilochaetus sp. and
surrounding coelomocytes. However, Ste- parasitic copepods are encysted by Sabella
phenson (1930) has contended that adult penicillum and S. spallanzanii (Dales and
stages were not attacked and only the cysts Dixon, 1981). A number of protozoan
were encapsulated, with the trophozoites species, particularly gregarines and coc-
within the cyst remaining unaffected by the cidians, are common parasites within the
process. Coelomocyte capsules formed coelom. Immature sporozoites were encap-
around nematodes and gregarines in L. ter- sulated while mature stages were not (Sied-
restris were described by Metchnikoff as licki, 1903). Among the cirratulid poly-
being composed of two layers: an inner, chaetes, live trophozoites of some parasitic
non-cellular and possibly chitinous layer, species were encapsulated while in other
which he believed to be secreted by the species only dead parasites were attacked
parasite, and an outer cellular layer, com- (Caullery and Mesnil, 1898).
posed of flattened and occasionally pig-
mented coelomocytes. Wound healing: response to injury
Cameron (1932) observed the successive Injuries to polychaetes, earthworms and
stages of capsule formation around cotton leeches may have greatly differing out-
threads which he had inserted into the coe- comes, depending on the nature and sever-
lomic cavity of lumbricid worms. An initial ity of the wound. This is due not only to
layer of coelomocytes surrounded the differences in morphology but also to dif-
thread, followed by the addition of new cell ferences in regenerative capacities of the
layers and a concurrent flattening of the three annelid groups. Earthworms are
inner layers. At the end of seven days, the highly compartmentalized animals with
capsules were invested by thin fibrous well-developed septa, so that they may sur-
strands and appeared to be partially cal- vive relatively severe wounds with minimal
cified. Accompanying the encapsulation loss of body fluids. In polychaetes such as
process, Cameron observed proliferation Arenicola or Terebella, punctures in the main
of the adjacent coelomic epithelium, with body of the worm may be fatal; the coelom
the cells growing outward from the coe- is generally continuous in this area, and
JSJ E. A. STEIN AND E. L. COOPER

aggregated coelomocytes. The fluid phase

of annelid blood and coelomic fluid does
not coagulate, except for that of a few
megascolecid oligochaetes (Benham, 1901;
Ohuye, 1937). Instead, coelomocytes
aggregate together to form a "wound plug"
which provides a mechanical barrier to fur-

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ther fluid loss (Cuenot, 1898; Chapron,
1970; Burke, 1974a). Muscle contraction
of the body wall around the wound also
serves to close off the opening (Burke,
1974a). Eight to twelve hours after wound-
ing, the plug possesses a layered appear-
ance: an outer surface layer of loosely asso-
ciated cells and cell fragments, a middle
layer of densely packed, flattened coelomo-
cytes with their long axes parallel to the
wound, and an inner layer of coelomocytes
elongated toward the wound (Burke,
1974a). The surface of the wound plug
forms a stratum over which, at approxi-
mately 12 hr, the epidermal cells start to
migrate, progressively covering the wound
with a single-celled layer of columnar epi-
thelium. In most cases, the surface of the
wound is completely covered by epithelium
FIG. 2. Electron micrograph showing diapedesis by at 32 to 48 hr. Body-wall tissue damaged
neutrophil (GC) of L. terrestris through the squamous by injury is either shed externally or
epithelium (SEp) of the coelomic cavity into the mus- removed by phagocytosis. Coelomocytes
cle layers of the body wall. Muscle fiber (MF); con-
nective tissue (CT). X6.000. (Reproduced with per- phagocytose much of the debris; however,
mission from Linthicum el at, 1977a.) ultrastructural studies have indicated that
epidermal columnar and basal cells may
also ingest muscle cell fragments (Burke,
1974ft).
contractions of the body wall tend to pump
out body fluids (Dales, 1978). Regenerative Shallow wounds in the body wall are also
abilities are also high in earthworms, and filled, within a few hours, by a provisional
loss of segments posterior to the clitellum wound covering consisting of coelomo-
(frequently occurring spontaneously by cytes and epidermal basal cells (Cameron,
autotomy after severe wounding) is usually 1932; Burke, 1974a, ft). Burke (1974ft) has
followed by growth of new segments (Cam- suggested, on ultrastructural evidence, that
eron, 1932). In polychaetes, only the most the basal cell layer of the epidermis may
posterior segments are easily renewed, and be derived from the same cell lineage as
leeches possess even more restricted regen- coelomocytes. Cameron (1932), using
erative capacities. worms in which coelomocytes had been
Wound healing has been observed both labelled by previous injections with India
by light microscopy (Cameron, 1932; Lieb- ink, observed that coelomocytes (primarily
mann, 1943; Herlant-Meewis and Deligne, hyaline amoebocytes) started to accumu-
1964; Clark, 1965) and electron micros- late within the coelom, underneath the
copy (Chapron, 1970; Burke, 1974a, b). In injured site, by 1 hr after wounding. Two
earthworms and polychaetes, wounds hours later, small areas of nearby coelomic
which penetrate the body wall into the coe- epithelium had started to proliferate. Six
lom are usually sealed off by a plug of hours post-wounding, coelomocytes had
 ANNELID INFLAMMATORY RESPONSES
begun to migrate into the body wall near
the injury and by 24 hr, a plug of coelo-
mocytes extended from the coelom into
the wounded area. The mass of cells con-
tained not only hyaline and granular amoe-
bocytes but also spindle-shaped cells,
believed to be undifferentiated cells of non-
coelomic origin. Subsequently (at 2-5 days),
epithelium grew over the surface of the
wound and muscle fibers were observed
spreading inwards from the edges of the
wound. Numerous fibrils (possibly connec-
tive tissue) were present and many small
blood vessels were growing inwards from
the periphery. By 10 days, healing was
nearly complete and tissues of the body
wall virtually normal in structure.
From such studies it is clear that leuko-
cyte infiltration is a major component of
annelid inflammatory responses. Addi-
tional evidence of leukocyte participation
in wound repair comes from ultrastruc-
tural studies of body wall grafts in L. ter-
restris and E. foetida, in which xenografts,
autografts and sham-operated (wounded)
controls were compared. Generalized
inflammatory reactions occurred after all
three procedures, beginning with diapi-
desis of coelomocytes through the coelo-
mic epithelium into the muscle layers (Fig.
2), leading to infiltration of coelomocytes
into the wounded area or into the grafts.
Although autografts healed without
destruction of the graft, moderate num-
bers of coelomocytes, identified as neutro-
phils, had entered the graft tissue and bed,
phagocytizing debris and damaged cells.
Xenografts were always destroyed, with
neutrophils that had invaded the graft tis-
sue actively phagocytosing viable muscle
tissue. In this latter instance, the leuko-
cytes appeared to be capable of an immu-
nospecific response in addition to a purely
inflammatory one (Linthicum et al., 1977;
Valembois and Roch, 1977).
In polychaetes, as in earthworms, initial
wound closure, especially in posterior seg-
ments, occurs as a result of muscle con-
traction around the wound combined with
formation of a plug of aggregated coelo-
mocytes (Dales, 1978). In Nephtys sp., Clark
(1965) observed that after tail amputation,
151
coelomocytes migrated to the wound site,
but damaged tissue was removed by fixed
phagocytes found around the nephridia and
parapodial blood vessels. In Owenia fusifor-
mis, loss of the anterior end of the body
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was followed by formation of a wound plug
by coelomocytes and subsequent over-
growth of the plug by a layer of epidermis.
Damaged tissue in Owenia was phagocy-
tosed by coelomocytes which subsequently
accumulated in the scar tissue, in healthy
tissue, or in the coelom under the wound
(Coulon and Thouveny, 1974). Although
instrumental in early stages of inflamma-
tion and wound healing, coelomocytes did
not participate directly in regeneration;
such cells or tissues were derived instead
from established tissues lying adjacent to
the injured area (Herlant-Meewis and van
Damme, 1964; Clark, 1965).
In leeches, muscle contraction around
the injured area is of primary importance
in closing ofF the wound and reducing fluid
loss (Sawyer and Fitzgerald, 1981). How-
ever, shallow wounds may be sealed by cells
that resemble the phagocytic cells of the
nephridial capsules, and which start to
migrate toward the area within an hour
after injury (LeGore and Sparks, 1971).
Although the healing process in leeches is
poorly understood, amoebocytes partici-
pate in phagocytosis and removal of dam-
aged tissue (Myers, 1935; LeGore and
Sparks, 1971).
HUMORAL RESPONSES
Inflammation in vertebrates is associated
with an extensive array of humoral fac-
tors—histamine, kinins, complement frag-
ments, prostaglandins and lysosomal
enzymes, to name but a few. Only a limited
number of such factors have been identi-
fied in annelids, and in these instances their
functions are poorly understood. Since in
most cases we are lacking specific infor-
mation, we will consider first the few sub-
stances that are known from vertebrate
studies to be related to inflammation, then
discuss those more general factors that are
related to inflammation by virtue of their
being produced in response to injury or
infection, or because they are known to
152 E. A. STEIN AND E. L. COOPER

TABLE 2. Humoral factors in annelid inflammatory respojises.

Annelid class
Humoral factors Polychaeta Oligochaeta Hinidinea References
Histamine, kalli- Not studied Present, but no in- Not studied Lemmi et al., 1973a, b.
krein, histidine crease with
decarboxylase wounding or im-

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mune challenge
Lysosomal en- Present, with vari- Present; with vari- Not studied Jollesandjuili, 1960;
zymes able increase after able increase after Schubert and Mes-
bacterial challenge wounding and im- sner, 1971; Valem-
mune challenge; bois, 1971; Stang-
no excretion by L. Voss, 1971; Stein
terrestris coelomo- and Cooper, 1978;
cytes during Dales and Dixon,
phagocytosis 1980; Marks et al.,
1981; Stein et al.,
1982.
Hemolysins, Both present Both present, both Hemagglutinins DuPasquier and Du-
hemagglu- increase after in- present prat, 1968; Cha-
tinins jection of erythro- teaureynaud-Duprat,
cytes in L. terrestris 1971; Cooper el al.,
1974; Gold and
Balding, 1975; Carte
and Russell, 1976;
Roch, 1979; Ander-
son, 1980; Stein et
al., 1980; Roch et
al., 1981a, b; Stein et
al, 1981; Valembois
elal, 1982.
Bacterial aggluti- Present Present Not studied Stein et al., 1981; An-
nins derson and Chain,
1982.
Antibacterial Present Present Not studied Roch elal., 1981a, b;
substances Lassegues et al.,
1981; Valembois et
al., 1982; Anderson
and Chain, 1982

interact with inflammatory agents (Table common occurrence during phagocytosis

2). or stimulation by non-phagocytosable sur-
faces containing immune complexes
Histamine and kinins ("frustrated phagocytosis") (Bainton,
Histamine, kallikrein and histidine 1980). Such phagocytosis-associated
decarboxylase normally occur in whole enzyme release has also been reported in
body extracts of the earthworms Lumbricus various invertebrate species, particularly
terrestris and Eisenia foetida (Lemmi et al., those of molluscs (Cheng, 1981). Acid
1973a). Inflammatory stimuli consisting of phosphatase and lysozyme have been
body wall grafts (autografts and xeno- detected in several species of polychaetes
grafts) and injections of sheep erythrocytes (Jolles and Juili, 1960; Schubert and Mess-
did not, however, result in a significant ner, 1971; Dales and Dixon, 1980) while
increase in the levels of any of the three acid phosphatase, lysozyme and /3-glu-
substances in either species of worm curonidase have been found in earth-
(Lemmi et al., 1973b). worms (Schubert and Messner, 1971;
Stang-Voss, 1971; Valembois, 1971; Stein
Lysosomal enzymes and Cooper, 1978; Marks et al, 1981).
The extracellular release of lysosomal Coelomocytes and coelomic fluid of the
enzymes by vertebrate phagocytic cells is a polychaetes Arenicola marina, Neoamphitrite
 ANNELID INFLAMMATORY RESPONSES 153

figulus and Nereis diversicolor normally con-
tain acid phosphatase and lysozyme, but
the coelomic fluid did not have any anti-
bacterial activity against those bacterial
species that were tested (Dales and Dixon,
1980). If bacteria were injected into worms,
activity by prior exposure to antigen have
proven unsuccessful (Anderson, 1980). In
the earthworms L. terrestris and E. foetida,
however, an increase in agglutinins (Coo-
per et al., 1974; Stein et al., 1980; Cha-
teaureynaud et al., 1981) and lysins (Cha-

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lysozyme and acid phosphatase levels rose teaureynaud-Duprat, 1971; Cooper et al.,
in some instances but were too variable to 1974) occurs after injection with erythro-
be statistically significant. However, when cytes of certain vertebrate species. The
coelomic fluid and cells of Nereis were in- increase in agglutinins of L. terrestris is not
cubated with bacteria in vitro, acid phos- specific to erythrocytes, but also occurs
phatase levels rose after 6 hr in both fluid after the injection of bovine serum albu-
and coelomocytes. min, several carbohydrates, amino acids
Coelomocytes and coelomic fluid of and a nucleotide. Saline injection does not
Lumbricus terrestris also contain acid phos- elicit the response, however (Wojdani et al,
phatase (Marks et al, 1981) as well as 1982). Both naturally occurring hemag-
/3-glucuronidase (Stein and Cooper, 1978) glutinins, and those produced after eryth-
with coelomocyte enzyme levels being rocyte injection, cross-react with bacteria,
highest in basophils and neutrophils (Stein agglutinating six of the twelve bacterial
and Cooper, 1978). Measurements of acid strains tested (Stein et al, 1981).
phosphatase released by Lumbricus coelo-
mocytes during in vitro phagocytosis of yeast Antibacterial substances
indicated that almost none of the enzyme Anderson and Chain (1982) have
was extracellularly secreted, even though reported that the polychaete, Glycera
coelomocytes avidly phagocytosed the yeast dibranciata, possesses a natural antibacte-
(Stein el al, 1982). rial substance active against a number of
Gram-negative but not Gram-positive bac-
Agglutinins and lysins teria. The antibacterial factor was a heat-
Various annelid species possess aggluti- labile protein, requiring Ca++ for activity,
5
nins a n d / o r lysins (Du Pasquier and and above 1 X 10 molecular weight. It was
Duprat, 1968; Cooper et al., 1974; Gold bactericidal but not bacterilytic, and hence
and Balding, 1975; Garte and Russell, 1976; not related to lysozyme.
Roch, 1979; Anderson, 1980; Stein et al, Coelomic fluid of the earthworm, L. ter-
1980; Chateaureynaud et al., 1981; Stein restris had no antibacterial activity when
etal, 1981), and although the relationship tested against a group of seven different
of these substances to inflammatory or bacterial strains (Cooper et al., 1969). How-
immune responses is, for the most part, ever, coelomic fluid of Eisenia foetida con-
unclear, they are presumed to have tains naturally occurring factors that are
immune-related functions (Anderson, both hemolytic (DuPasquier and Duprat,
1980). They are structurally unrelated to 1969) and antibacterial (Roch et al., 1981 b).
vertebrate immunoglobulins and vary from The factors were lytic for the erythrocytes
species to species in their molecular struc- of a number of vertebrate species and pre-
ture and biochemical composition (Gold vented the growth of 6 of 23 bacterial
and Balding, 1975; Roch et al., 1981a). In strains. Sensitive bacterial strains were both
most instances, they have been tested for Gram-positive and negative. The factors
activity against erythrocytes and have were also present in the fluid of developing
therefore been classified as hemagglutinins cocoons of Eisenia (Valembois et al., 1982).
and hemolysins (Cooper et al., 1974; Garte When coelomic fluid was separated by
and Russell, 1976; Anderson, 1980) but in polycrylamide gel filtration, the antibac-
several species (Eisenia foetida and Lumbri- terial substances were present in two bands
cus terrestris) have also exhibited activity of approximately 4X 104 and 4.5 X 104
against bacteria (Roch et al., 19816; Stein molecular weight (Roch et al, 19816). By
et al., 1981; Valembois et al., 1982). In isoelectric focusing, they have been sepa-
polychaetes, attempts to increase their rated into 4 isoforms, with all worms con-
154 E. A. STEIN AND E. L. COOPER

taining 3 of the 4 forms (Roch et al., 1979).
The bacterial strains sensitive to the fac-
tors are pathogenic for Eisenia, and were
shown to have a surface antigen in com-
mon with sheep erythrocytes (Lasseques et
al., 1981). The antibacterial factors were
Burke.J. M. 1974a. Wound healing in Eisenia foe tida
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Burke, J. M. 1974ft. Wound healing in Eisenia foe tida
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epidermal tissues. Cell Tiss. Res. 154:83-102.

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heat-sensitive proteins with associated car- Cameron, G. R. 1932. Inflammation in earthworms.
bohydrate and lipid groups. J. Gen. Path. Biol. 35:833-872.
Caullery, M. and F. Mesnil. 1899. Sur les parasites
CONCLUDING REMARKS internes des annelides polychetes, en particulier
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2, 28:491-496.
and 2, summarizing information currently Chapron, C. 1970. Etude histologique, infrastruc-
available on inflammatory processes in turale et experimentale de la regeneration cepha-
annelids, that our information is severely lique chez le lombricien, Eisenia foetida. Ann.
limited. Although certain components of Embr. Morph. 3:235-250.
the response, such as phagocytosis, encap- Chateaureynaud-Duprat, P. 1971. Etude des reac-
tions de defense de nature humorale chez le lom-
sulation and wound healing are somewhat bricien Eisenia foetida. C.R. Acad. Sci. Paris. 273:
better understood, other areas are defi- 1647-1649.
cient or lacking even in a descriptive way. Chateaureynaud, P., J. Lestage, and F. Izoard. 1981.
Do annelids possess cells analogous to mast Some functions of Lumbricus leukocytes in pro-
cells and basophils, and do vasoactive sub- tection mechanisms. In J. P. Solomon (ed.), Aspects
of developmental and comparative immunology, pp.
stances participate in the response? Do 11 — 16. Pergamon Press, New York.
feedback mechanisms operate between the Cheng, T. C. 1981. Bivalves. In N. A. Ratcliffe and
various leukocyte types during an inflam- A. F. Rowley (eds.), Invertebrate blood cells, Vol.
matory response? What specific chemotac- 1, pp. 233-300. Academic Press, New York.
tic factors are present and how do they Clark, M. E. 1965. Cellular aspects of regeneration
in the polychaete Nephtys. In V. Kiortis and H.
exert their effect? The entire area of anne- A. L. Trampusch (eds.), Regeneration in animals,
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Metchnikoffs time. We hope that new Hall, New Jersey.
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ACKNOWLEDGMENT 14:402-406.
Partially supported by NIH Grants Cooper, E. L., C. A. Lemmi, and T. C. Moore. 1974.
AI15976-03, HD 09333 and NSF Grant Agglutinins and cellular immunity in earth-
PCM 82-04879. worms. Ann. N.Y. Acad. Sci. 234:34-50.
Cooper, E. L. and E. A. Stein. 1981. Oligochaetes.
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