AMER. ZOOL., 23:205-211 (1983)
Inflammatory Reactions of the Protochordata1
RICHARD K. WRIGHT AND EDWIN L. COOPER
Department of Anatomy, School of Medicine, Center for the Health Sciences,
University of California, Los Angeles, California 90024
SYNOPSIS. Ascidian inflammatory responses are characterized by an influx of blood cells
into areas containing foreign material or injured tissue. The sequestering and removal of
foreign substances is a function of their chemical nature and size. Small particulate materials are removed by phagocytosis; substances too large to be phagocytosed are encapsulated. Blood cells involved in inflammatory reactions are small undifFerentiated cells
(hemoblasts, lymphocytes), hyaline and granular leucocytes and morula cells.
INTRODUCTION
Inflammation occurs in living tissue when
it is injured or invaded by foreign materials
after which, fluid and blood cells accumulate at the injured site. Inflammatory
responses accomplish two things: 1)
removal of foreign substances and 2) disposal of damaged tissue followed by healing. All kinds of infection (bacteria, fungi,
parasites) and tissue damage (wounds, tissue grafting) elicit inflammatory responses.
These responses may be acute, lasting several hours or days, or chronic, lasting much
longer. Sequestering and elimination of
foreign substances depends upon their
nature and size. Small particulate materials
are effectively removed by phagocytosis. If
the offending substances are too large to
be phagocytosed or the irritant persists for
prolonged periods, granulation tissue forms
around the material encapsulating it.
Protochordates comprise the subphyla
Cephalochordata and Urochordata of the
phylum Chordata. At some time in their
life cycle, protochordates possess the three
distinguishing characteristics of chordates:
a notochord, dorsal tubular nerve cord and
pharyngeal gill slits. Cephalochordates,
commonly known as Amphioxus, are small,
translucent, free-living, filter-feeding
marine animals found buried in sand on
the sea bottom in many parts of the world.
Urochordates, commonly referred to as
tunicates or ascidians, are also filter-feeding marine animals with a world-wide dis-
tribution. Their larval stages are free-living whereas adults are generally sessile;
pelagic species, specialized for a planktonic
existence, however, do exist. As a group,
urochordates are divided into three classes:
Ascidiacea, Thaliacea and Larvacea. The
Ascidiacea contain the majority of species
and are the most common and typical tunicates.
The means by which cephalochordates
protect themselves from infectious agents
are unknown. There have been no studies
on their internal defense system. They have
no circulating blood cells (Moller and Philpott, 1973) and the presence of wandering
phagocytic cells within their body tissues is
uncertain. Within the urochordates, information is lacking for the Thaliacea and
Larvacea. Thus, the main focus of this
review will be on the inflammatory
responses of the Ascidiacea.
ASCIDIAN ANATOMY
The Ascidiacea are the most generalized
class of tunicates and believed to be the
stock from which the other tunicate classes
and vertebrates evolved (Berrill, 1955).
Their external surface is a special mantle
called the tunic or test. It serves as an effective mechanical barrier against microorganisms found in the environment. The
tunic is usually thick, varies in consistency
from gelatinous to fibrous, and is composed of cellulose, protein and inorganic
compounds. Within the tunic, there are two
distinct body regions: 1) an anterior pharyngeal region containing the pharynx
' From the Symposium on Comparative Aspects of
(branchial basket) and 2) an abdominal
Inflammation presented at the Annual Meeting of the region containing primarily the digestive
American Society of Zoologists, 27-30 December
tract, gonads and heart.
1981, at Dallas, Texas.
205
206
R. K. W R I G H T AND E. L. COOPF.R
TABLE 1. Total blood cell counts and differentials in adult ascidian blood.*
Species
Ascidia atra
Ascidia ceratodes
Ascidia nigra
Ciona intestinalis
Halocynthia
aurantium
Halocynthia roretzi
Perophora viridis
Pyura mirabilis
Pyura stolonifera
Styela clava
Styela plicala
Mogula manhattensis
Number of
hemocytes
per ml of
blood
7
(X 10 )
—
4.7-14.5
3.2-7.9
0.8-1.4
0.9-1.3
0.7-2.7
1.0-1.4
1.1-1.7
—
1.0-1.4
1.8-6.8
0.3-0.5
0.6-1.0
1.7-2.5
% hemoblasts
(lymphocytes)
% leucocytes
(hyaline/
granular)
<1
15
81
3-5
<1
17
22
5
<1
<1
<1
<1
5
<1
—
3
4
27
46
60
42-52
17-22
6-17
12-17
3
42-52
80
6
94
43
30
34
40-70
54-77
76-87
75-85
90
40-50
15
88
1
13
2
—
2-4
2
—
<1
2
<1
—
3
:
vacuolated
cells
: other cell
types
* For references, see Wright, 1981.
The circulatory system consists of a
tubular heart enclosed in a pericardium.
The heart contains no valves and consists
of a single layer of fusiform myoepithelial
cells whose basal portion contains a bundle
of cross striated myofibrils. Blood is
pumped through the system by means of
peristaltic contractions originating at one
end of the heart. Blood flow and the direction of peristalsis reverses periodically due
to the presence of two pacemakers, one at
each end of the heart. A single blood vessel
exits from each end of the heart.
True blood vessels are lacking, and the
vascular system is essentially a hemocoel
(Berrill, 1950) with blood flowing into various sinuses or lacunae in the connective
tissue. The lacunae are not lined with an
endothelium except at each end of the heart
where there is a loose reticulum of cells
lining the large vessels (Millar, 1953). Principal channels have a connective tissue lining, one cell thick. Tunic vessels are lined
by an epithelium consisting of a single layer
of large, columnar epidermal cells. Blood
cells can be observed migrating across the
vessel walls, penetrating the tunic substance.
Circulating through this network of
channels is a blood plasma and various types
of blood cells. The plasma is colorless
although in some species it may appear colored due to the presence of various pigments within the blood cells. Ascidian
plasma does not clot, is iso-osmotic with
sea water, slightly alkaline, acid or neutral
in pH and contains a low concentration of
proteins. It has a low carbon dioxide capacity and oxygen concentrations are similar
to those of sea water. No specialized respiratory carriers for oxygen are present.
Ascidian blood contains six to nine different cell types that exhibit considerable
variation in different species. All blood cells
are actively amoeboid and are not confined
to the vascular system. Blood cells move
freely from the blood spaces into the connective tissues and tunic where they can be
found randomly distributed or in large
concentrations. The percentage of blood
occupied by blood cells is on the order of
l-27c (Endean, 1955; Vallee, 1967). Blood
cell morphologies have been described for
over 70 species (reviewed by Wright, 1981).
While the terminology used by different
investigators is not always the same, the
various cell types can be classified into several categories: 1) undifferentiated cells
(hemoblasts or lymphocytes), 2) leucocytes
(hyaline or granular), 3) vacuolated cells
(signet ring, compartment or morula), 4)
various pigmented cells and 5) nephrocytes. Their proportions within the blood
cell population show considerable differences among species (Table 1). In general,
undifferentiated cells occur in low percentages, leucocytes at intermediate levels
and vacuolated cells predominate.
PROTOCHORDATE INFLAMMATORY REACTIONS
Ascidian blood cells are a renewing cell
population (Ermak, 1975). They have a
rapid proliferation rate which, at the steady
state, is balanced by the rate of cell loss.
Cell renewal occurs in the circulating blood
and in hemopoietic tissues located principally in the pharynx, around the digestive
tract and, as distinct nodules in the body
wall of advanced species (Millar, 1953;
Ermak, 1976).
ASCIDIAN INFLAMMATORY REACTIONS
Hemocytes involved
Undifferentiated cells (hemoblasts or
lymphocytes), the various types of leucocytes (hyaline and granular) and vacuolated cells (predominantly the morula cell)
are involved in inflammatory reactions.
The particular cell type(s) eliciting the
response depend upon the nature of the
foreign material introduced into the animal.
Small, undifferentiated cells containing
no granules or vacuoles have traditionally
been called lymphocytes (Peres, 1943;
Freeman, 1964; Overton, 1966; Ermak,
1975). Studies recognizing the role of these
cells as stem cells have, more appropriately, designated them hemoblasts (Peres,
1943; Mukai and Watanabe, 1976; Ermak,
1976; Milanesi and Burighel, 1978). Lymphocytes have been distinguished from
hemoblasts on the basis of size and nuclear
morphology. Hemoblasts are larger, have
a distinctive large nucleolus and little chromatin whereas lymphocytes have no
nucleolus and the chromatin occurs in
patches along the inner surface of the
nuclear membrane and in the interior of
the nucleus. Both have a narrow ring of
finely granular basophilic cytoplasm containing several small, round mitochondria,
a Golgi apparatus, a few cisternae of rough
endoplasmic reticulum and abundant free
ribosomes. Conventional histochemical
staining indicates the presence of cytoplasmic proteins and carbohydrates but no
lipids or glycogen. Both exhibit amoeboid
activity, producing pseudopodia, but are
not phagocytic. While it is possible that the
hemoblast and lymphocyte are different cell
types, it remains to be demonstrated experimentally. Both have been observed to be
207
present in inflammatory foci. Their specific roles in inflammation however, have
not been defined.
Leucocytes consist of two cell types: hyaline and granular. They are frequently
referred to as amoebocytes, granulocytes,
macrophages and phagocytes. They are
actively amoeboid and phagocytic. Hyaline
leucocytes have a homogeneous cytoplasm
filled with granules of uniform size, a welldeveloped Golgi complex, rough endoplasmic reticulum and mitochondria. A few
vacuoles containing granular inclusions
may also be present. Histochemical staining reveals the presence of cytoplasmic
protein, diffuse periodic Schiff (PAS) positive material, carbohydrate, a positive oxidase material but no lipids or acid mucopolysaccharides. Granular leucocytes have
a coarsely granular cytoplasm containing
distinct oval or spherical granules with
refractive properties. The granules are
homogeneous, strongly electron dense and
with conventional blood stains, may be acidophilic, basophilic or neutrophilic. They
give positive oxidase and PAS reactions and
contain protein. Lipid droplets and masses
of dense particles characteristic of glycogen are also present.
Vacuolated cells consist of three cell
types: signet ring, compartment and morula
cells. They characteristically contain one
or more large vacuoles and appear to be
interrelated, passing through a progressive
cycle from signet ring to compartment to
morula (Endean, 1960; Kalk, 1963). During the course of their development, they
accumulate and concentrate heavy metals,
forming a heavy metal-protein complex
within the vacuoles. Morula cells have a
berry-like appearance and have been called
green cells, vanadocytes and ferrocytes, the
latter two names derived from the fact that
in some species, they contain vanadium, or,
in others, iron. In living cells, the vacuoles
appear colorless to yellow-green; in fixed
cells, the vacuoles contain electron dense
material that may be homogeneous, granular or filamentous and is PAS positive.
Morula cells are the predominant cell type
found within the tunic matrix and their
primary function appears to be tunic formation. It has been suggested that they
208
R. K. WRIGHT AND E. L. COOPER
TABLE 2. Ascidian inflammatory reactions.
Species
Agent inducing inflammation
Type of reaction
Ciona intestinalis
Cynthia papillosa
Halocynthia aurantium
Phallusia mammillata
Tunic injury
Leucocyte infiltration and
tunic repair
Ciona intestinalis
Tunic transplants
Leucocyte infiltration and
graft rejection
Halocynthia pyriformis
Unknown
Marine hydrozoans
Wooden splinters
Bacteria
Vertebrate erythrocytes
Carmen gruel
Carbon particles
Carmine particles
Vertebrate erythrocytes
Glass fragments
Parasitic copepods
Parasitic copepods
Parasitic copepods
Leucocyte infiltration
Ascidia mentula
Ciona intestinalis
Distaplia unigermis
Halocynthia aurantium
Mogula manhattensis
Ciona intestinalis
Mogula manhattensis
Ascidiella aspersa
Microcosmus savignyi
Styela gibbsi
may secrete the tunic (Endean, 1960), be
instrumental in the organization of the
tunic (Saint-Hilaire, 1931; Das, 1936) or
function in both capacities (Smith, 1970).
Morula cells also appear to be involved in
the encapsulation of foreign objects too
large to be phagocytized.
Phagocytosis and clearance
Encapsulation (granulomas)
Rinehart et al, 1981a, b) and agglutinins
that may have a biological role in inflammatory reactions.
Phagocytosis and clearance
Small paniculate foreign materials
introduced into the tunic or vascular system are phagocytosed by the hyaline and
Leucocyte infiltration
granular leucocytes (Table 2). Vertebrate
The introduction of foreign materials or erythrocytes (Wright, 1974; Parrinello et
tissue injury is followed by a pronounced al, 1977), colloidal carbon (Smith, 1970;
emigration of large numbers of blood cells Parrinello et al, 1977) or bacteria (Metch(hemocytes) into the affected area (Table nikoff, 1892;Cantacuzene, 1919; Thomas,
2). This emigration is accomplished- by 1931) injected into the tunic are phagohemocyte diapedesis through the walls of cytized and cleared from the tunic matrix
blood channels. What stimulates the hemo- usually within 48 hr. Similar results have
cytes to emigrate is unknown. While che- been obtained after injections of carmine
motactic factors are probably involved, no particles (Saint-Hilaire, 1931; Ivanovastudies to demonstrate their existence have Kazas, 1966; Anderson, 1971), trypan blue
been undertaken in ascidians. In addition, (Anderson, 1971), colloidal thorium dioxthe various plasma mediator factors (ki- ide suspensions (Brown and Davies, 1971)
nins, complement, clotting factors) and tis- or vertebrate erythrocytes (Wright, 1974)
sue mediator factors (vasoactive amines, into the vascular system or peritoneal cavprostaglandins, lysosomal components, ity.
leukocytosis factors, etc.) known to be
Phagocytosis involves recognition. How
involved in mammalian inflammatory ascidian hyaline and granular leucocytes
responses have yet to be investigated in recognize foreign material is unknown.
tunicates. Ascidian plasma and tissues how- Opsonization by a factor(s) may be necesever, are known to contain bactericidins sary and the natural agglutinins present in
(Johnson and Chapman, 1970), anti-bac- the plasma and tissue fluids may function
terial, viral and fungal factors (Cheng and in this capacity. Direct evidence for aggluRinehart, 1978; Carter and Rinehart, 1978; tinin involvement is lacking. These agglu-
PROTOCHORDATE INFLAMMATORY REACTIONS
tinins however, appear to be produced by
one of the blood cell types (Wright and
Cooper, 1981) and probably have some
function related to phagocytosis. After recognition and attachment of the foreign
material to the leucocyte surface, the material is internalized into vacuoles. Elimination of the engulfed particles depends on
their chemical nature. Intracellular degradation by lysosomal enzymes probably
occurs against organic materials. Histochemical examination to confirm the presence of lysosomal enzymes however, is
lacking. Inert substances that cannot be
degraded appear to be transported and
released into the external environment
through the branchial sac (Anderson,
1971), tunic (Saint-Hilaire, 1931; IvanovaKazas, 1966), intestine (Brown and Davies,
1971) and neural gland (Peres, 1943;
Godeaux, 1964; Ivanova-Kazas, 1966).
Encapsulation, granuloma formation
Foreign materials too large to be phagocytosed are encapsulated (Table 2). The
response is confined to the tunic and does
not appear to occur in the vascular system
or perivisceral cavity. Encapsulation
responses are a type of chronic inflammation characterized by the accumulation
of blood cells around the object and the
formation of a compact capsule. The reaction is locally confined to the area containing the foreign object and is similar to the
formation of vertebrate granulomas.
Naturally occurring encapsulation responses have been observed against commensal and parasitic copepods (Breciana
and Liitzen, 1960; Monniot, 1963; Dudley,
1968) and have been experimentally
induced by inserting splinters (Metchnikoff, 1892) and glass fragments (Anderson,
1971) or by injecting bacteria (Thomas,
1931) and vertebrate erythrocytes (Wright,
1974; Wright and Cooper, 1975; ParrineWoetai, 1976, 1977) into the tunic. With
bacteria, there is an initial phagocytosis followed by necrosis at the injection site. The
borders of the necrotic region are then surrounded by concentric rings of cells, effectively walling off the inflamed area. Glass
fragments inserted into branchial tissue
results in the formation of multilayered
209
structures made up of morula cells and
strands of tunicin produced by the morula
cells.
The injection of high concentrations of
human, duck, rabbit or sheep erythrocytes
into the tunic produces an initial agglutinated mass. A whitish halo containing
granular material and vacuolated cells surrounds the agglutinated mass within 48 hr
after injection. Capsule formation is usually complete by 6-10 days. Within 2-3 wk,
a large gelatinous or liquid blister forms
within the capsule. The overlying tunic
becomes thinner and ruptures, producing
a wound in the tunic. The capsule and its
contents are then released to the external
environment. Following capsule ejection,
the tunic usually heals.
Tunic wounds and tissue grafts
Tunic wounds readily provoke inflammatory reactions characterized by: 1)
morula cell infiltration, 2) morula cell cytolysis which releases intracellular products
that fill the wound edges and 3) subsequent
wound healing and tunic regeneration.
Incisions in the tunic of Halocynthia aurantium cause an initial migration and congregation of morula cells at the wound edge
(Smith, 1970). Although hyaline and granular leucocytes are present in the injured
area, they do not appear to increase in
numbers. After several days, the matrix
surrounding the wound becomes filled with
material released from disintegrating
morula cells and there is a concurrent
pinching together and coalescence of the
wound edges. Twenty days after injury, the
external edge of the wound has been overlaid with a dense layer of cuticle. The
fibrous material below the wound is lifted
towards the wound and constricts there.
The wound scar is denser and not as heavily
pigmented as uninjured tunic. Injury to the
tunic of Pyura stolonifera is also followed by
an influx of morula cells into the damaged
region at the sea water interface, morula
cell cytolysis and release of fiber-producing
intraglobular material (Endean, 1955).
Tissue transplants also provoke inflammatory responses, the type of response
dependent upon the nature of the tissue
transplanted. Allogeneic tunic transplants
210
R. K. WRIGHT AND E. L. COOPER
INTRODUCTION
OF
FOREIGN KATEHIAL
?» TISSUE I I W
LEUCOCYTE INFILTRATION
INTO PPEA
PHAGOCYTOSIS AHD
ENCAFJUIATIQN
CLEARANCE
rGRANULOl1A
FIG. 1. Summary of the sequence of events occurring in ascidian inflammatory responses.
healing and tissue repair to occur. With
foreign substances, their sequestering and
removal is a function of their nature and
size. Small particulate materials are phagocytosed by hyaline and granular leucocytes. In contrast, morula cells surround
and encapsulate larger objects. Encapsulation probably represents an extension of
the primary function of morula cells, tunic
formation. After phagocytosis or encapsulation, the offending materials are
removed, allowing tissue repair.
From the foregoing, it is obvious that we
have not progressed beyond the descriptive phases of ascidian inflammatory reactions. Many questions remain unanswered.
What stimulates the blood cells to migrate
to injured areas? Are various plasma and
tissue mediators involved? How do ascidian
blood cells recognize foreign material and
subsequently degrade the material? With
answers to these questions, we will obtain
a better understanding of ascidian inflammatory reactions, especially as models for
explaining the evolutionary development
of inflammation.
in Ciona intestinalis heal to the graft bed
after five to seven days. During an eight
week period following grafting, a persistent inflammatory response characterized
by increased numbers of undifferentiated
cells (lymphocytes), granular leucocytes and
phagocytic cells occurs at the interface of
host and graft tissue. No increase in morula
cells was observed. Autografts showed no
increase in the number of undifferentiated
cells. The ultimate fate of the grafts however, is unknown due to increased animal
mortality and termination of the study at
the end of eight weeks (Reddy et al., 1975).
In contrast to tunic grafts, branchial sac
ACKNOWLEDGMENTS
auto- and allogeneic grafts do not fuse with
Supported by NIH Grant HD 09333-06.
host tissues in Mogula manhattensis (Anderson, 1971). The grafts become infiltrated
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CONCLUDING REMARKS
The sequence of events occurring in
ascidian inflammatory responses are summarized in Figure 1. When tissues are
injured, either mechanically or by the presence of foreign materials, there is a pronounced leucocyte infiltration into the area.
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