Download Cellular Receptors and Signal Transduction in Molluscan

INTEGR. COMP. BIOL., 43:305–312 (2003)
Cellular Receptors and Signal Transduction in Molluscan Hemocytes: Connections with the
Innate Immune System of Vertebrates1
JUDITH E. HUMPHRIES
AND
TIMOTHY P. YOSHINO2
Department of Pathobiological Sciences, School of Vet Medicine, University of Wisconsin-Madison, 2115 Observatory Drive
(Biotron), Madison, Wisconsin 53706-1087
SYNOPSIS. The involvement of circulating hemocytes as the principal cellular effector mediating molluscan
immune responses is well established. They participate in a variety of internal defense-related activities
including microbial phagocytosis, multicellular encapsulation, and cell-mediated cytotoxicity reactions that
are presumed to be initiated through foreign ligand binding to hemocyte receptors and subsequent transduction of the binding signal through the cell resulting in appropriate (or in some cases, inappropriate)
hemocyte responses. At present, however, although functional evidence abounds as to the existence of hemocyte ‘‘recognition’’ receptors, few have been characterized at the molecular level. Similarly, signal transduction systems associated with various receptor-mediated hemocyte functions in molluscs are only beginning to be investigated and understood. This review examines what is currently known about the molluscan
hemocyte receptors and the putative signal transduction pathways involved in regulating their cellular behaviors/activities. The cumulative data implies the presence of various hemocyte-associated receptors capable
of binding specific carbohydrates, extracellular matrix proteins, growth factors, hormones, and cytokines.
Moreover, receptor-ligand interactions appear to involve signaling molecules similar to those already recognized in vertebrate immunocyte signal transduction pathways, such as protein kinases A and C, focal
adhesion kinase, Src, Ca21 and mitogen-activated protein kinase. Overall, the experimental evidence suggests
that molluscan immune responses rely on molecules that share homology with those of vertebrate signaling
systems. As more information regarding the molecular nature of hemocyte recognition receptors and their
associated signaling molecules is accumulated, a clearer picture of how hemocyte immune responses to
invading organisms are regulated will begin to emerge.
INTRODUCTION
A common theme that integrates the vast field of
immunology is the critical ability of cells comprising
the immune system of each organism to communicate
with both their internal and external environments.
‘‘Communication,’’ in this context, is taken to mean
being able to receive a chemical signal(s), usually via
surface membrane receptors, transducing that signal
across the membrane and through the cytoplasm, eventually to target effector molecules that, in turn, trigger
appropriate responses (Heldin and Purton, 1996). In
cells associated with the vertebrate immune system,
receptor-ligand interactions resulting in the regulation
(i.e., activation or deactivation) of numerous cellular
functions have been shown to involve a complex network of intracellular signaling molecules. For example, cellular adhesion and motility, cytotoxic responses, endocytosis, induction of cell death, and the like,
have been linked to signal transduction pathways involving a diversity of cellular proteins including, but
not restricted to, various small guanine nucleotide
binding proteins (GTPases) such as Rho and Ras,
phosphoinositide 3- and Src-kinases, protein kinases C
or A (PKC/PKA) and members of the mitogen-activated protein kinase (MAPK) family including erks
and p38 (Dong et al., 2001; Greenberg, 2001; Torgersen et al., 2002).
In contrast to the extensive data on immunocyte signaling systems found in higher vertebrates, relatively
little is known about such systems in invertebrates,
especially what molecules may be involved in signal
transduction pathways and whether different cellular
functions are regulated through the same or different
pathways. For molluscs the mechanisms of signal
transduction, in general, have been derived mainly
from neurobiological studies (Crow et al., 1998; Nakhost et al., 1998; Tensen et al., 1998; Tierney, 2001)
that have begun to establish the presence of intracellular signaling pathways, many of which appear to be
homologous with those of mammals. In this review we
will focus on the current knowledge of the identity and
role of signaling molecules and pathways involved in
molluscan immune responses, with emphasis on the
gastropods. The discussion of cell signaling will be
presented according to the type of cellular receptors
believed to initiate such signals in the primary immune
effector cells, the hemocytes.
GASTROPOD HEMOCYTES REPRESENT A MOLECULARLY
HETEROGENEOUS POPULATION
Circulating blood cells known as hemocytes, represent the main cellular component of the molluscan
immune system. Several studies examining the composition of hemocyte populations of gastropods suggest that hemocytes are composed of a mixture of different subpopulations of cells. Flow cytometric analyses of hemocytes from the freshwater snails Biomphalaria glabrata (Johnston and Yoshino, 2001) and
Planorbarius corneus (Franceschi et al., 1991) dem-
1 From the Symposium Comparative Immunology presented at the
Annual Meeting of the Society for Integrative and Comparative Biology, 2–6 January 2002, at Anaheim, California.
2 Corresponding author; E-mail: [email protected]
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onstrate that circulating hemocyte populations could
be divided into two main subtypes differing in their
granularity and size. In both molluscan species, the
larger granular cell subpopulation also has been shown
to differ functionally from the smaller agranular cell
type. For example, only the small round hemocyte
(RH) of P. corneus are able to lyse a human cell line
in a natural killer (NK) cytotoxicity test indicating an
early sharing of functional characteristics between RH
and NK-related cells (Franceschi et al., 1991). Large
granular hemocytes of B. glabrata, characterized by
the absence of the monoclonal antibody BGH1 surface
marker (Yoshino and Granath, 1985; Johnston and
Yoshino, 2001), is highly phagocytic compared to the
nonphagocytic BGH11 agranular cell subset. A similar
distinction of hemocyte subpopulations based on expression of a unique surface epitope also has been
made in Lymnaea stagnalis; namely hemocytes expressing the monoclonal antibody LS1 surface marker
are thought to represent undifferentiated cells (LS11
subset), whereas the LS12 cell subset is comprised of
mature hemocytes (Dikkeboom et al., 1985). It is presumed that hemocyte subpopulations that differ both
chemically and functionally are regulated in their activities or behaviors through specific receptors and the
signals conveyed by their interaction with appropriate
ligands. Unfortunately, in comparison with immunocytes of vertebrates, little is known about the receptors
mediating immune-related activities or their associated
pathways of intracellular signaling in molluscan hemocytes.
HEMOCYTE RECEPTORS
The first step in executing an immune response is
the detection by hemocytes of foreign invaders and/or
non-self cells/tissues, presumably through receptors
associated with the surface membrane. Signals generated by ligand binding are then transduced across the
membrane, usually in conjunction with receptor phosphorylation, triggering a cascade of downstream chemical reactions, ultimately directing these signals to target organelles (e.g., nucleus, cytoskeleton) resulting in
the induction of appropriate cellular responses (Heldin
and Purton, 1996). A number of receptors from hemocytes of gastropod molluscs have been identified
based on crossreactivity with various anti-receptor antibodies or through functional assays, although relatively few have been characterized at the molecular
level. For the purpose of discussion here, hemocyte
receptors are grouped into several broad categories:
Lectins (or lectin-like receptors), integrin-related proteins, and growth factor/hormone/cytokine-like receptors. Many of these receptors have been described or
identified in cells from B. glabrata, the intermediate
host of the human blood fluke Schistosoma mansoni,
and therefore, will be the focus of this review. However, this is a rapidly evolving field of study and one
should not ignore an important body of literature covering aspects of molecular reception and signaling in
other nonhemocytic molluscan systems, especially
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T. P. YOSHINO
those involving neuronal control of digestion/feeding
(e.g., Canesi et al., 2000; Furukawa et al., 2001) and
reproductive behavior (Kendel, 2001; Tierney, 2001).
HEMOCYTE LECTINS AND THEIR SIGNALING NETWORKS
It has been established that numerous carbohydrate
(CHO)-binding proteins are present in cell-free hemolymph or plasma of B. glabrata (Monroy et al.,
1992; Mansour et al., 1995). The finding that some of
these proteins are able to bind to parasite larval stages
has prompted speculation that they may be functionally-associated with host immunity. Recently, a unique
family of proteins with CHO-reactivity, referred to as
fibrinogen-related proteins or Freps (Adema et al.,
1997; Leonard et al., 2001), has been shown to be
induced in snails in response to infection with the
trematode, Echinostoma paraensei, and specifically
precipitate excretory-secretory proteins released by
larvae. These lectin-like molecules are produced in hemocytes and appear to mediate their CHO-reactivity
through a calcium-dependent carbohydrate recognition
domain (CRD) located in the fibrinogen-like C-terminal segment of the protein (Adema et al., 1997). Complementary DNA encoding another B. glabrata-derived polypeptide with homology to a mammalian selectin C-type CRD also has been found (Duclermortier
et al., 1999). Selectins are a family of mammalian
CHO-reactive membrane proteins found on endothelial
cells, leukocytes and/or platelets that serve as adhesion
receptors in a variety of roles including leukocyte extravascular trafficking and inflammation (Kilpatrick,
2002; Patel et al., 2002). Like the Freps, the B. glabrata selectin-like polypeptide is synthesized in hemocytes and in cells of the B. glabrata embryonic
(Bge) cell line, and occurs primarily as a secreted (soluble) protein. At present, however, a functional role,
if any, of these soluble lectins in mediating specific
cell-cell adhesive functions or anti-parasite activity is
unknown.
In addition to hemocyte-derived soluble lectin-like
proteins, the presence of CHO-reactive receptors at the
surface of gastropod and bivalve hemocytes also has
been demonstrated through biochemical and functional
analyses (Vasta et al., 1982, 1984), direct binding studies using glycosylated bovine serum albumin (BSA)
(Hahn et al., 2000), or through CHO-specific inhibition of hemocyte functions such as phagocytosis
(Richards and Renwrantz, 1991; Fryer et al., 1989),
cellular adherence to parasites (Castillo and Yoshino,
2002) or binding to larval excretory-secretory glycoproteins (Johnston and Yoshino, 2001). However, despite a considerable body of functional evidence for
the existence of membrane-associated hemocyte lectinlike receptors in molluscs, to date such receptors have
yet to be isolated and molecularly characterized. Consequently, very few studies of specific signaling pathways associated with lectin-like receptors in these cellular systems have been undertaken.
Phagocytosis, or the adherence and internalization
of small foreign particles (e.g., bacteria) is normally
RECEPTORS
AND
SIGNALING
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MOLLUSCAN HEMOCYTES
307
FIG. 1. Combined phase-contrast/epifluorescence photomicrographs of Biomphalaria glabrata hemocytes (A) and a cell of the B. glabrata
embryonic (Bge) cell line (B) phagocytosing fluoresceinated Echerichia coli. Phagocytosed bacteria (green) are indicated by arrows.
initiated by the binding of particles to specific cell receptors. Such adhesive interactions may be the result
of particles binding directly to recognition receptors at
the cell membrane, or indirectly, through the reactivity
of phagocyte receptors with particle-bound soluble
recognition molecules (opsonins). The signaling pathways regulating phagocytosis in mammalian cell systems are well delineated, especially those associated
with Fc, complement, Toll-like and lectin receptors
(Aderem and Underhill, 1999; Kwiatkowska and Sobota, 1999; Hebert, 2000; Greenberg and Grinstein,
2002; Termeer et al., 2002 and others). Given that endocytosis is an ‘‘ancient’’ function shared by cells of
all protozoan and metazoan phyla, it may be assumed
that a high degree of conservation also exists in the
general signaling pathways mediating this common
cellular activity. However, with the exception of recent
studies in insects, very little is known regarding
phagocytosis signaling pathways in most invertebrate
groups, including molluscs. For example, studies by
Foukas et al. (1998) have demonstrated that phagocytosis of E. coli by Drosophila hemocytes appears to
require Ras and mitogen-activated protein kinase
(MAPK) activation for bacterial attachment and the
involvement of a ß-integrin for internalization. Focal
adhesion kinase (FAK) phosphorylation and Src-kinase also appear to be important in bacterial phagocytosis (Metheniti et al., 2001). Recently identified
transmembrane peptidoglycan recognition proteins,
PGRP-SA (Michel et al., 2001) and PGRP-LC (Gottar
et al., 2002; Ramet et al., 2002), exhibiting binding
specificities for gram-positive and gram-negative bacterial peptidoglycans, respectively, appear to confer resistance to bacterial infection, in part through a protective phagocytic response. Interestingly, PGRP-SA
utilizes the Toll signaling network, whereas PGRP-LC
receptor involves the IMD-Relish intracellular signaling pathway. Although the above studies did not address whether or not bacterial phagocytosis might in-
volve possible lectin-like receptors, the fact that lipopolysaccharides or peptidoglycans represent major
binding ligands mediating both the phagocytic and signaling events leaves open this possibility.
Preliminary studies in molluscs using B. glabrata
hemocytes and cells of the B. glabrata embryonic
(Bge) cell line have shown that these cells also are
able to phagocytose heat killed E. coli (Fig. 1), and
that such activity is inhibited in a dose-dependent fashion by calphostin C, a specific inhibitor of diacylglycerol (DAG)-dependent PKC (Fig. 2). These results
suggest that phagocytosis of E. coli by snail hemocytes
may occur through activation of a PKC-associated signaling pathway. However, Bge cells appeared to be
more sensitive to calphostin C than hemocytes, being
significantly inhibited in their phagocytic activity at 1
nM calphostin C compared to controls, and to a level
of phagocytosis comparable to hemocytes at 10 nM of
drug (Fig. 2). This result may be due in part to a higher
variability in in vitro phagocytosis rates in hemocytes,
or possibly to a more complex network of signaling
pathways associated with hemocyte receptors mediating bacterial phagocytosis. Alternative pathways appear to be used in other molluscan species. For example, phagocytosis by oyster hemocytes is altered by
noradrenaline through a ß-adrenergic receptor-coupled
cAMP/PKA pathway, similar to that found in vertebrate systems (Lacoste et al., 2001). Clearly further
study of signaling molecules regulating phagocytosis,
especially those mediated by membrane-associated
lectins (Fryer et al., 1989; Richards and Renwrantz,
1991), within and across molluscan classes is warranted.
Hemocytic encapsulation responses to metazoan
parasites, in contrast to bacterial phagocytosis, require
the coordinated interactions of hemocytes, not only
with the parasite and its secreted products, but also
with each other, to form multicellular capsules that effectively eliminate the foreign intruder (Yoshino et al.,
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J. E. HUMPHRIES
FIG. 2. Effects of Biomphalaria glabrata hemocyte (A) and Bge
cell (B) treatments with varying concentrations of calphostin C, an
inhibitor of diacylglycerol (DAG)-dependent protein kinase C
(PKC), on phagocytosis of fluoresceinated E. coli. Cells were simulataneously exposed to calphostin C and approx. 75 bacteria/snail
cell, followed by incubation at 228C for 2 hr. Snail cell/E. coli preparations were then washed in snail saline, treated with 1% trypan
blue to quench extracellular bacterial fluorescence and finally, fixed
with 4% buffered paraformalin. 100–200 cells/sample were evaluated as positive or negative for phagocytosis. Raw percentage data
were analyzed by one-way ANOVA and means comparisons by the
Dunnett’s post hoc test. For graphic purposes, however, data are
shown as % reduction relative to the CBSS saline control (set at
100%). The number of independent replicates for each control/treatment are indicated in parentheses below each data bar.
2001). For example, hemocytes from a S. mansoniresistant strain of B. glabrata would be required to
migrate to the vicinity of invading larvae (sporocyst
stage), initiate binding to the sporocyst tegument (recognition), produce a multicellular capsule around the
parasite through cell-cell interactions, and finally effect
a ‘‘killing’’ event involving cytotoxic activation. The
specific receptors mediating different phases of this antiparasitic response have not yet been identified, although recent studies have implicated the involvement
of different complex carbohydrates in the initial binding of B. glabrata hemocytes and Bge cells to live
sporocysts (Castillo and Yoshino, 2002), representing
the ‘‘recognition’’ phase of the parasite-host interaction, and in the triggering of hemocyte cytotoxic reactions (Hahn et al., 2001). In this latter case, exposure
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T. P. YOSHINO
of B. glabrata hemocytes to specific BSA-conjugates
of mannose, galactose or fucose stimulated cellular
production of reactive oxygen species (ROS) such as
superoxide or hydroperoxides, thus implicating hemocyte lectin-type receptors in signaling a potential
sporocyst killing response (Hahn et al., 2000). Previous findings that oyster hemocyte ROS are inhibited
by noradrenalin, and can be modulated by ß-adrenoreceptor agonists and antagonists, suggest that ß-adrenergic-type receptors also may be involved in regulating ROS production in some molluscan species (Lacoste et al., 2001). Likewise, exposure of bivalve hemocytes to phorbol myristate acetate (PMA), a
diacylglycerol analogue, resulted in an increase in
ROS (Pipe, 1992; Anderson, 1994) suggesting the involvement of a PKC-type signal transduction mechanism in the generation of an oxidative burst. To date,
however, we still know very little regarding the specific signaling pathways triggered by receptor-CHO
binding or those involved in regulating ROS production in molluscan hemocytes.
Excretory-secretory glycoproteins (ESP), released
by early developing larval trematodes previously have
been reported to affect snail hemocyte behaviors such
as motility and cell spreading (Lodes and Yoshino,
1990; Noda and Loker, 1989a), phagocytosis (Noda
and Loker, 1989b; Connors and Yoshino, 1990; Nunez
et al., 1997), ROS production (Connors and Yoshino,
1990) and encapsulation responses (Loker et al.,
1992), suggesting that hemocyte receptor-ESP interactions were providing intracellular signals critical to
hemocyte immune function. Moreover, binding of ESP
to B. glabrata hemocytes appears to be through lectinlike CHO binding receptors (Hertel et al., 2000; Johnston and Yoshino, 2001), indicating the involvement
of lectin-like receptors in transducing these behaviormodulating cellular signals. In support of this notion,
Hertel et al. (2000) have shown that in response to E.
paraensei ESP, B. glabrata hemocytes display cell
rounding accompanied by intracellular calcium transients or fluxes. Interestingly, exposure of cells to a
mannose-BSA neoglycoconjugate produced a nearly
identical pattern of calcium transients and rounding as
echinostome ESP treatment, inferring that echinostome
ESP-mediated signaling may be initiated through lectin-like receptors and involve Ca12 mobilization as a
second messenger system. Important questions still remain to be answered when considering the complex
molecular interactions between hemocytes and their
larval trematodes: Can the binding of different CHO
ligands to hemocytes result in either up- or down-regulation of specific responses, and if so, is this the result
of receptor-ligand triggering of distinct intracellular
signal transduction pathways? Have some larval trematodes ‘‘discovered’’ (and are exploiting) signaling
pathways that lead to cellular deactivation, thus circumventing or ward off potentially damaging immune
reactions? These represent some of the very interesting, but challenging, questions to be addressed in future studies.
RECEPTORS
AND
HEMOCYTE INTEGRIN-RELATED RECEPTORS
SIGNALING NETWORKS
SIGNALING
AND
THEIR
As integrin receptors have been reported to regulate
vertebrate cell spreading and motility (Hynes, 1992;
Holly et al., 2000), the possibility that homologues of
this same family of receptors might play a similar role
in molluscan cell behaviors was investigated. Initial
studies demonstrated that the tetrapeptide, arg-gly-aspser (RGDS), could inhibit the spreading activity of
both B. glabrata hemocytes and cells of the closelyrelated Bge cell line, suggesting the presence of an
integrin-like receptor(s) and their involvement in cellular adhesion/spreading behavior (Davids and Yoshino, 1998). A ß-integrin receptor subunit cDNA subsequently was cloned from Bge cells and a near identical partial sequence also was identified in B. glabrata
hemocytes (Davids et al., 1999; Yoshino et al., 1999).
To examine which signaling molecules might be involved downstream of receptor activation, a panel of
specific inhibitory drugs targeted to various signaling
molecules were used in in vitro cell spreading assays.
The results of drug inhibition studies demonstrated
that proteins with pharmacological similarities to PKC,
Ras and MAPK kinase (Mek) may be regulating both
Bge cell and hemocyte spreading (Humphries et al.,
2001; J.E.H., unpublished observations). However,
compared to Bge cells, a lower sensitivity of B. glabrata hemocytes to specific PKC/Ras/MAPK inhibitors (unpublished observations), suggests that RGDSmediated spreading in these cells may be regulated by
a ‘‘network’’ of signaling pathways possibly involving
multiple receptors, rather than a single, predominant
one. At the molecular level, evidence in B. glabrata
for specific homologues of mammalian signaling molecules is scant. An intracellular receptor for activated
protein kinase C (RACK) has been identified from
whole snail cDNA, and was shown to be capable of
binding activated mammalian PKC (Lardans et al.,
1998). This finding provides indirect evidence for the
presence of a PKC-like protein(s) in this snail species.
In addition, we have cloned from Bge cells a partial
cDNA sequence with significant homology to p38
(Accession no. AF486290), a MAPK family member
associated with stress (inflammatory) signaling (Huang
and Erikson, 1996). As more information is gained
regarding the types of signaling molecules present in
snail cells, a clearer picture of the potentially complex
networks involved in simple adhesive/spreading behaviors of hemocytes should begin to emerge.
HEMOCYTE GROWTH FACTOR/HORMONE/CYTOKINE
RECEPTORS AND INTRACELLULAR SIGNALING
Because cytokines, growth factors and hormones are
known to play prominent roles in regulating cellular
activity in vertebrate immunity, it has been of interest,
from a comparative and phylogenetic perspective, to
investigate whether or not such molecules elicit responses in molluscan hemocytes, presumably through
binding interactions with appropriate receptors. Sev-
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MOLLUSCAN HEMOCYTES
309
eral studies have revealed the ability of circulating hemocytes to respond to a variety of soluble vertebrate
factors, indirectly suggesting the presence of receptors
to a variety of growth, hormonal and cytokine-like factors. For example, receptors for platelet-derived
growth factor (PDGF-alpha/ß) and transforming
growth factor-ß (TGF-ß) have been reported in hemocytes of mussels Mytilus galloprovincialis, and
when bound to their respective ligands, appear to modulate various cellular functions such as phagocytosis
and cell motility (Ottaviani et al., 1997a; Kletsas et
al., 1998). Exposure of Haliotis (abalone) hemocytes
to epidermal growth factor (EGF) or porcine insulin
stimulates protein and DNA synthesis (Lebel et al.,
1996), again suggesting the presence of their respective receptors and the ability to transduce ligand-mediated intracellular signaling. An insulin-like receptors
cDNA has been cloned from the Bge cells line (Lardans et al., 2001) and adrenocorticotrophic hormone
(ACTH)-like receptor mRNA was localized in Mytilus
hemocytes (Ottaviani et al., 1998), thus supporting the
prediction of the presence of growth factor/hormone
receptors in molluscan hemocytes. Similarly, peptide
derivatives of proopiomelanocortin (POMC), which
include ß-endorphin, ACTH and alpha-melanotropin
(melanocyte-stimulating hormone; alpha-MSH), have
been shown to affect hemocyte behavior in a variety
of molluscan species, implying the presence of their
hemocyte-associated peptide receptors (Stefano et al.,
1989; Duvaux-Miret et al., 1992; Sassi et al., 1998).
The presence of cytokine-like receptors on molluscan
hemocytes, likewise, has been inferred by the induction of various hemocyte functions by exposure to vertebrate cytokines. For example exposure of B. glabrata
hemocytes to human recombinant interleukin (IL)-1
results in the priming for superoxide production and
increased phagocytosis (Granath et al., 1994; Connors
et al., 1995), while IL-8 induces in mussel hemocytes
increased phagocytic activity and a chemotactic response (Ottaviani et al., 2000).
The direct ‘‘cause and effect’’ relationship illustrated above between ligand treatment and hemocyte reactivity to ligand exposure suggests not only the presence of appropriate receptors, but also the ability to
communicate or transduce the ligand-initiated signal
into the cell to produce an effector response. These
results, of course, raise the question: what is the nature
of the signaling pathways being used in these systems?
Based on pharmacological studies, as observed with
integrin-like receptors, by employing specific inhibitor
drugs it appears that ACTH-receptor signaling induces
Mytilus hemocyte shape changes though an adenylate
cyclase/cAMP/ protein kinase A (PKA) pathway (Sassi et al., 1998). In contrast, corticotropin-releasing hormone (CRH) appears to induce cellular shape changes,
not only through activation of PKA and PKC, but also
requires extracellular calcium and the synergistic action of cAMP and inositol 1,4,5 triphosphate (Malagoli
et al., 2000). The cell shape changes induced by
PDGF-alpha/ß and TGF-ß in Mytilus hemocytes ap-
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J. E. HUMPHRIES
pear to be transduced through a PKA/PKC phosphoinositide-phospholipase C-type pathway similar to the
CRH-receptor (Ottaviani et al., 1999). However,
PDGF signaling, unlike that of TGF, is extracellular
calcium-independent (Ottaviani et al., 1997b). Little is
known regarding cytokine receptor-mediated signaling
in molluscan hemocytes. Pharmacological studies on
mussel hemocytes indicate that IL-8-induced changes
in cell spreading may be regulated through PKC and
PKA, and is associated with reorganization of actin
filaments. The involvement of the common signaling
elements PKC and PKA by a variety of receptor types
(growth factors, hormones, cytokines, integrins) suggest a conservation of signaling pathways within molluscan hemocytes. However, more research in this area
is needed, especially at the molecular level, to begin
to more precisely define all of the molecules involved
in linking receptors to cell function.
CONCLUSIONS
As described in this review, molluscs possess a very
effective, multifaceted immune/defense system, incorporating both cellular and humoral components. By its
nature, an immune system depends on the ability of
cells to recognize the presence of foreign antigens (by
pattern recognition) and elicit appropriate responses.
The entire process of ‘‘recognition-to-response’’ depends on complex intracellular signal transduction systems that we are only beginning to understand in the
context of molluscan immunity. Although research of
this area in molluscs is still in its infancy, recent discoveries to date imply that molluscan immune-mediated signal transduction is well developed and most
likely utilizes a variety of molecular pathways shared
in common with vertebrates and the more intensely
studied invertebrates such as Drosophila. In general
terms, hemocyte-mediated immune responses of molluscs reflect those innate cellular responses of vertebrates; they involve surface membrane receptors including integrin- and lectin-like proteins, as well as
receptors capable of binding mammalian cytokines,
growth factors and peptide hormones. Similarly, there
is evidence to suggest that protein kinases such as
PKA, PKC, FAK and MAPK, small G proteins such
as Ras, and second messengers like cAMP and [Ca12]i,
are participants in regulating molluscan hemocyte
functions such as cell spreading, phagocytosis or ROS
production. However, much of the available data on
which this review was based, had been acquired
through functional and/or inhibition assays, as well as
immunocytochemistry utilizing specific, but crossreactive, antibody probes. Clearly their exists a need for
more research at the molecular level in order to fully
characterize homologous receptors, signaling molecules and pathways involved in hemocyte immune responses in molluscs.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the assistance
of Courtney Copeland, supported by the University of
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T. P. YOSHINO
Wisconsin SRP-Biology Undergraduate program, and
Michelle Browne in generating some of the preliminary data on B. glabrata phagocytosis presented in this
paper.
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