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First published online March 17, 2006
Journal of Experimental Biology 209, 1326-1335 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02118
Interaction between non-specific electrostatic forces and humoral factors in haemocyte attachment and encapsulation in the edible cockle, Cerastoderma edule
School of Environment and Society, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, UK
* Author for correspondence (e-mail: e.c.wootton{at}swansea.ac.uk)
Accepted 23 January 2006
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Summary |
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 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
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Key words: invertebrate immunity, encapsulation, cell attachment, surface charge, electrostatic force, humoral factor, haemocyte, bivalve, Cerastoderma edule
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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).
In general, a capsule of haemocytes encloses the foreign body and cytotoxic
products, such as degradative enzymes and free radicals, are released by the
haemocytes in an attempt to destroy the invader. This immune defence reaction
has been most extensively studied in insects and research has shown this
process to be highly complex (Ratcliffe,
1993), involving a diversity of cellular and molecular processes
(e.g. Cheng and Garrabrant,
1977; Ouaissi et al.,
1990; Nappi et al.,
1995; Loret and Strand,
1998; Choi et al.,
2002; Whitten et al.,
2004). Physicochemical properties such as surface charge and
hydrophobicity also influence parasite–haemocyte interactions (e.g.
Walter and Williams, 1967;
Lackie, 1983;
Lackie, 1986;
Lavine and Strand, 2001);
however, these factors have received less attention than the highly specific
biochemical and molecular mechanisms.
Since the 1920s, scientists have shown considerable interest in the
biological relevance of surface charge within vertebrate systems
(Mehrishi and Bauer, 2002),
and there is much evidence to support a role for surface charge in
immunological-based reactions. For example, sialic acids, which are present on
leucocytes to help prevent non-specific interactions between cells, are
removed upon cellular activation during early `non-self' recognition,
resulting in a decreased cell surface negative charge and unmasking of further
cell receptors and ligands. This increases cellular interactions and leads to
effective immune defence (Rieu et al.,
1992; Kelm and Schauer,
1997; Crocker and Varki,
2001). Such a reaction highlights the synergistic action of
non-specific electrostatic forces and highly specific receptor–ligand
interactions.
Electrostatic forces are also implicated in antibody-antigen interactions
involving complement and major histocompatibility complex (MHC)
(Morikis and Lambris, 2004a;
Morikis and Lambris, 2004b),
and cationic antimicrobial peptides exploit their positive surface charge in
order to interact with anionic lipids of microorganism membranes
(Zasloff, 2002). In addition,
parasites and pathogens use their negative surface charge to help avoid host
immune responses (Crocker and Varki,
2001). Surface charge also has a pivotal role in the development
of gene/drug/antigen delivery systems to target macrophages, which may aid
treatment of globally important diseases such as schistosomiasis, cancer, HIV
and tuberculosis (Ashan et al., 2002).
In invertebrates, cell surface charge has generally been implicated in
host–parasite interactions (e.g.
Saraiva et al., 1989;
Monteiro et al., 1998;
Akaki et al., 2001;
Souto-Padrón, 2002),
and in multicellular parasites, surface charge has been largely investigated
with respect to the encapsulation response. In insects, chromatography beads
and thread are used to mimic parasites (e.g.
Vinson, 1974;
Dunphy and Nolan, 1980;
Ratner and Vinson, 1983;
Paskewitz and Riehle, 1994;
Lavine and Strand, 2001),
probably because they lack the common pathogen-associated molecular patterns
(PAMPs; e.g. lipopolysaccharide, peptidoglycan and ß-1,3-glucan). The
absence of all parasite-associated molecules from synthetic targets allows the
basic mechanism of encapsulation, involving non-specific physicochemical
properties and electrostatic forces, to be studied. This mechanism is
potentially common to all host–parasite interactions, and provides the
basis on which highly specific biochemical interactions are imposed. Studies
using beads have generally shown that positively charged beads are
encapsulated most vigorously (e.g. Walter
and Williams, 1967; Vinson,
1974; Dunphy and Nolan,
1980; Lackie,
1983; Ratner and Vinson,
1983), but few detailed investigations justify on the role of
electrostatic forces and their interaction with opsonic humoral factors in
invertebrate immunity.
One extensively studied multicellular parasite system in invertebrates is
the trematode blood fluke, Schistosoma mansoni and its intermediate
host, the gastropod mollusc, Biomphalaria glabrata (see reviews by
Bayne et al., 2001;
Loker and Bayne, 2001;
Yoshino et al., 2001). Marine
bivalve molluscs are also common hosts to multicellular parasites, including
trematodes (de Montaudouin et al.,
2000); however, information on host resistance mechanisms to such
parasites is very limited, despite the growing aquaculture industry and the
high commercial value of molluscan shellfish
(FAO, 2005). Cheng and Rifkin
(Cheng and Rifkin, 1970), who
recognised the high prevalence of metazoan parasites in marine bivalves,
examined the host response to these parasites histologically, and proposed
five different types of encapsulation. Research, however, has not continued
into understanding the dynamics of capsule formation in bivalve molluscs.
The present study investigates the non-specific basic mechanisms involved in cell attachment and encapsulation in the edible cockle, Cerastoderma edule. Synthetic beads and thread were used to mimic the inert and inactive metacercarial cysts of the trematode, Himasthla sp., commonly found encapsulated in the foot of C. edule. The investigation describes how non-specific electrostatic forces and humoral plasma factors interact to mediate haemocytic immune responses.
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Materials and methods |
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Observations of trematode capsules
Squash preparations and histology were used to confirm that C.
edule uses haemocytic encapsulation as an immune defence reaction. The
cockle foot was examined for metacercarial cysts of Himasthla spp.,
parasitic trematodes of C. edule (e.g.
Wegeberg et al., 1999;
de Montaudouin et al., 2000)
commonly found in this tissue. For squash preparations, the cockle foot was
dissected longitudinally, and each section squashed between two glass slides
until Himasthla sp. cysts were clearly visible under a dissecting
microscope. Standard paraffin wax sections were also used to examine the
structure of the haemocytic capsule surrounding Himasthla sp. cysts.
The cockle foot was stored in an excess volume of Bouin's seawater fixative,
double wax embedded (Humarson,
1979), sectioned and then stained with Cole's Haematoxylin and
Eosin.
In vivo and in vitro cell attachment and encapsulation experiments
Factors involved in mediating haemocyte attachment and encapsulation were
studied, both in vivo and in vitro, using chromatography
beads of various matrices, charges and functional groups, and Nylon
monofilament (thread) as encapsulation targets
(Table 1). Trematode
metacercarial cysts were not used in encapsulation studies as they could not
be removed intact from host tissues. Prior to use, beads were washed 10x
in 0.05 mol l–1 Tris-buffered saline (TBS; 0.05 mol
l–1 Tris/HCl containing 2% NaCl, pH 7.4) and resuspended to
produce a 15% v/v stock solution of beads/TBS. The beads ranged in size
(Table 1), which prevented the
use of precise haemocyte:bead ratios in the experiments. Bead preparations
were stored at 4°C and then vortexed immediately before use. Nylon thread
was cleaned with detergent, rinsed 5x with distilled water followed by
70% ethanol, before final washing with ultrapure water. The thread was air
dried and stored in sterile tubes at room temperature.
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Characterization of encapsulation
The encapsulation response varies greatly from one invertebrate species to
another and may or may not involve the formation of multi-layers of flattened
haemocytes (see Discussion for details). The encapsulation response of C.
edule to synthetic beads, both in vivo and in vitro,
involves the attachment of haemocytes to the beads to mediate their
aggregation, without the formation of classical multi-layered capsules. For
clarity and quantification, this reaction is designated encapsulation and
justification for this is provided in the Discussion.
For the present study, the C. edule encapsulation process is divided into two different stages. The first stage is identified as the onset of encapsulation, during which haemocytes first recognise and attach to beads, both in vivo and in vitro (Fig. 1A). The second stage is the end-point (or completion) of encapsulation, characterized by the formation of large haemocyte/bead aggregates and by the lack of further incorporation of haemocytes or beads into these structures (Fig. 1B). In addition, in vivo, the end-point could be identified by the formation of aggregates containing >100 beads, which prevented any outflow of beads from the dissected foot. The time difference between these two stages was recorded and reflected the rate of the encapsulation response.
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With nylon thread, the first stage (or onset) of encapsulation was identified as the attachment of individual haemocytes and the second stage (or end-point) was characterized as the attachment of large haemocyte clumps or the formation of a haemocytic sheath around the thread.
The times investigated to determine the rate of encapsulation of targets, in vivo, were 10 min, 30 min, at hourly intervals up to 6 h, at 12, 18, 24, 48 h, or until no further response was observed. In vitro, times were as for in vivo but only up to 24 h. For each target, the time taken for the two stages of encapsulation to be reached was recorded.
In vivo experiments
Bead suspension (100 µl of 15% v/v beads/TBS) was injected into the
proximal region of the cockle foot, using a 25-gauge needle. The foot was then
removed and placed in 0.05 mol l–1 TBS. The tissue was
dissected and the recovered beads added to 500 µl Baker's formol calcium
(4% formaldehyde, 1% calcium chloride, 2% NaCl) in sterile 24-well tissue
culture plates (Fisher Scientific UK, Leicestershire, UK) for subsequent
examination. Nylon thread was treated similarly, except that it was threaded
longitudinally though the foot of the cockle inside a 21-gauge needle, which
was removed to leave the thread in situ. Both beads and nylon thread
were examined using an inverted microscope and the haemocytic response
recorded.
In vitro experiments
The encapsulation response using whole haemolymph (i.e. haemocytes with
plasma) was compared with that of haemocytes suspended in 0.05 mol
l–1 TBS (i.e. haemocytes without plasma), thus also assessing
the role of humoral factors and opsonisation in cell attachment and
encapsulation.
For whole haemolymph samples, haemolymph (500 µl per individual) was
withdrawn from the posterior adductor muscle using a 26-gauge needle, into an
equal volume of ice-cold 0.05 mol l–1 TBS. For haemocyte
preparations suspended in 0.05 mol l–1 TBS (i.e. without
plasma), whole haemolymph was withdrawn, as above, into an equal volume of
ice-cold anticoagulant buffer (0.05 mol l–1 TBS containing
0.5% EDTA and 2% glucose, pH 6.2) (Pipe et
al., 1995). This was centrifuged at 55 g for 12
min at 4°C, the cell pellet washed once with anticoagulant buffer and then
resuspended in 1 ml 0.05 mol l–1 TBS. Haemocyte viability
tests were performed on all samples using both a dye exclusion assay (0.2%
Eosin in 0.05 mol l–1 TBS) and a cell viability kit (L-7013,
Molecular Probes Inc, Eugene, OR, USA). Results from both tests were
consistent with each other, so only the dye exclusion assay was used
subsequently. Samples with a viability of <90% were always rejected.
Sterile tissue culture plates (24-well, Fisher Scientific UK) were coated with 1% ECM gel (Sigma-Aldrich Company Ltd, Dorset, UK) in 0.05 mol l–1 TBS for 1 h at room temperature to prevent haemocytes from attaching to the bottom of the wells. The wells were washed 5x with 0.05 mol l–1 TBS, haemocyte samples (with or without plasma) from each individual were added to duplicate wells (400 µl well–1; 1.21±0.81x106 cells well–1) and 10 µl of 15% v/v beads/TBS solution (ca. 1500 beads) or five pieces of Nylon thread (5 mm in length) added to each well. The plate was incubated at 9°C on a rocking platform and the wells examined for encapsulation at the designated time intervals. Cell viability was monitored throughout the experiment.
Role of surface charge and plasma in cell attachment and encapsulation, in vitro
Comparisons were made between living and dead haemocytes, both in the
presence and absence of plasma, towards beads of original, and then reversed,
surface charge, in order to establish the role of electrostatic forces and
humoral opsonic factors in cell attachment and encapsulation.
The surface charge of beads was reversed in order to investigate the role of electrostatic forces. The positive surface charge of DEAE Sepharose beads was reversed to negative by binding heparin, poly-L-glutamate or poly-L-aspartate to the bead surface. Stock bead solution was incubated with 100 units ml–1 heparin or 1 mg ml–1 poly-L-glutamate/poly-L-aspartate for 16 h at 9°C with agitation. Alternatively, negatively charged, CM Sepharose beads were incubated with 1 mg ml–1 poly-L-lysine in order to reverse their surface charge to positive. After incubation, all beads were washed 3x with 0.05 mol l–1 TBS and confirmation of binding, and thus reversal of surface charge, was determined using FITC-poly-L-lysine labelling (see below).
Dead haemocyte preparations were incorporated into this experiment in order to eliminate cell attachment due to active cell signalling and `non-self' recognition processes of living cells. This allowed attachment to be investigated solely based on the surface properties of haemocytes and beads. Dead haemocyte preparations (both with and without plasma) were produced by incubating haemolymph samples (see above for preparation) at 15°C until haemocyte viability was zero (between 48–72 h).
The experimental set-up followed that of the in vitro experiments described above, with duplicate wells for each bead type and haemocyte preparation. Plates were incubated for 15 h at 9°C on a rocking platform and the encapsulation response towards each bead type recorded as either +++, ++, or + (see Table 2 for descriptions). Cell viability was also measured after 15 h incubation.
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The role of humoral opsonic factors was examined in two control experiments. First, foetal calf serum (FCS) was incorporated into experiments to act as a C. edule plasma control. Haemolymph from five animals (500 µl per individual) was withdrawn into ice-cold anticoagulant buffer and pooled on ice. Cell pellets were produced, as above, from three 1.5 ml portions and resuspended either in 1.5 ml cell-free haemolymph, FCS (200 µg FCS protein ml–1 TBS, i.e. equivalent cell-free haemolymph protein concentration) or TBS. The encapsulation of DEAE Sepharose beads (positively charged) or CM Sepharose beads (negatively charged) was then studied in vitro, as above, using the three haemocyte preparations. Second, DEAE and CM Sepharose beads were pre-incubated with either cell-free haemolymph, FCS or TBS overnight at 4°C with agitation. Beads were then washed 3 times with TBS. Encapsulation of these bead preparations was then studied in vitro using haemocytes without plasma (i.e. cells resuspended in TBS). For both control experiments, presence/absence of encapsulation was recorded for each well after a 15 h incubation.
Measurement of surface electronegativity
A modified method of Pendland and Boucias
(Pendland and Boucias, 1991)
was used to label negatively charged surfaces. The method uses binding of
FITC-labelled poly-L-lysine, a highly positively charged compound,
to surfaces to establish surface electronegativity. The effectiveness and
reliability of FITC-poly-L-lysine binding was initially confirmed
using encapsulation targets of known surface charge
(Table 1). Following this, the
surface electronegativity of all haemocyte preparations, modified beads and
Himasthla sp. cysts was measured. Samples were incubated with
FITC-poly-L-lysine (0.1 mg ml–1 in 0.05 mol
l–1 TBS) for 30 min at 9°C with agitation, washed
1x in 0.05 mol l–1 TBS and examined using a Zeiss
photomicroscope II (450–490 nm filter). Fluorescing samples represented
a negative surface charge.
Statistical analyses
For statistical analyses, data were first checked for normality using the
Kolmogorov–Smirnov test. Gaussian populations were analysed using a
one-way analysis of variance (ANOVA) with a Tukey's multiple comparison post
test, whilst non-Gaussian populations were either transformed to normality
(i.e. Gaussian) and analysed as above, or analysed using the non-parametric
equivalent (Prism, Graphpad Software Inc., San Diego, CA, USA).
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In vivo experiments
Times recorded for the onset and completion of encapsulation are presented
in Fig. 3A. Statistical
analyses revealed significant differences in the rate of encapsulation, i.e.
in both the onset and completion, between targets of different surface
charges, but not within targets of the same surface charge. Positively charged
targets (DEAE Sepharose and QAE Sephadex) were encapsulated significantly more
rapidly than both negatively charged (Nylon and CM Sepharose) and neutral
targets (Sepharose and Toyopearl) (P<0.001). In addition,
negatively charged targets were encapsulated significantly faster than neutral
ones (P<0.001).
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In the presence of plasma, positively charged targets were encapsulated most rapidly (P<0.001), followed by negatively charged ones, and then neutral targets, which were encapsulated least rapidly and to a lesser extent. In addition, in vitro encapsulation of neutral targets was noticeably lower when compared with the in vivo response (Fig. 3A). Only after 13.20±0.80 h (mean ± s.e.m.) and 13.80±0.92 h (mean ± s.e.m.), for Sepharose and Toyopearl beads, respectively, did individual haemocytes attach to beads, and no further progression of encapsulation was observed.
In the absence of plasma, haemocytes only attached to positively charged targets (DEAE Sepharose and QAE Sephadex). The response, however, was significantly slower than in the presence of plasma (P<0.001; Fig. 3B). In addition, unlike in the presence of plasma where haemocytes became flattened and spread upon attachment (Fig. 4A,C), haemocytes in the absence of plasma retained a rounded morphology even after attachment (Fig. 4B,D).
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Assessment of cell viability showed a decrease of 1.1±0.21% and 1.4±0.27% (mean ± s.e.m.) for haemocytes with and without plasma, respectively, after a 1 h incubation. This decreased further to 3.8±0.24% and 7.6±0.40%, after 5 h incubation, 8.7±0.48% and 16.8±1.0% after 10 h (by which time most encapsulation reactions were complete), 10.9±0.82% and 17.6±0.98% after 15 h, and finally 27.5±0.81% and 45.3±1.2% after 24 h.
Role of surface charge and plasma in cell attachment and encapsulation, in vitro
Results from this investigation are summarized in
Table 2. The extent of
encapsulation towards each target depended upon the surface charge of the
bead, whether or not plasma was present, and whether the haemocytes were alive
or dead.
With respect to surface charge, haemocytes from all preparations (i.e. in the presence and absence of plasma, living and dead) attached to all positively charged targets. The haemocytic response did not vary with different surface molecular patterns (Table 2). In contrast, haemocytes from only one preparation (living haemocytes in the presence of plasma) attached to negatively charged targets. Also, changing the bead surface charge from positive to negative [i.e. DEAE (+) to DEAE plus heparin (–)], resulted in decreased haemocyte attachment, whereas changing the bead surface charge from negative to positive [i.e. CM (–) to CM plus poly-L-lysine (+)] increased the attachment of cells. Reversal of DEAE Sepharose surface charge to negative, through binding of poly-L-glutamate and poly-L-aspartate, produced very similar encapsulation responses to that of heparin binding (data not shown).
The presence of plasma also strongly influenced haemocyte attachment to all bead types, including those with reversed surface charge. With live haemocytes, plasma resulted in a higher degree of cell attachment when compared with its absence. This was illustrated by haemocytes spreading on the bead surface, larger numbers of haemocytes attaching to beads, and by production of larger, more tightly bound, bead/haemocyte aggregates (see Table 2). In contrast, with dead haemocyte preparations, the presence of plasma lowered the degree of cell association with positively charged beads.
The role of humoral opsonic factors in the enhanced encapsulation response of living haemocytes in the presence of plasma was confirmed, since haemocytes resuspended in FCS did not encapsulate negatively charged CM Sepharose beads. Neither did haemocytes resuspended in TBS. In contrast, haemocytes resuspended in cell-free haemolymph encapsulated both DEAE Sepharose and CM Sepharose beads. The specificity of the response was further confirmed by haemocytes resuspended in TBS encapsulating CM Sepharose beads pre-incubated in cell-free haemolymph after 15 h incubation, but not beads pre-incubated in FCS or TBS. In contrast, DEAE Sepharose beads pre-incubated in either cell-free haemolymph, FCS or TBS were all encapsulated after 15 h.
Cell viability had decreased by 10.2±0.47% and 18.7±1.1%, for haemocytes in the presence and absence of plasma respectively, after the 15 h incubation.
Measurement of surface electronegativity
Fluorescence of negatively charged beads
(Fig. 5A), but not positively
charged or neutral ones, confirmed that FITC-poly-L-lysine only
bound to negatively charged surfaces. Both living and dead haemocytes in the
absence of plasma also fluoresced, confirming a negative surface charge
(82.41±1.46% and 85.39±1.68% (mean ± s.e.m.)
respectively, Fig. 5B).
Unfortunately, the presence of plasma precipitated
FITC-poly-L-lysine, therefore a negative surface charge could not
be confirmed for haemocytes in these preparations. Himasthla sp.
cysts were also negatively charged (Fig.
5C,D). In addition, FITC-poly-L-lysine binding
confirmed reversal of bead surface charge.
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). In
the present study, details of the encapsulation response of the edible cockle,
Cerastoderma edule, are described and detailed information is
revealed of a synergistic interaction between surface charge and humoral
plasma factors. This synergistic relationship is probably relevant to
encapsulation responses of other invertebrates groups. Encapsulation occurred in response to metacercarial cysts of the trematode, Himasthla sp. in the foot of C. edule; however, not all cysts were encapsulated. Seawater containing dye failed to penetrate Himasthla sp. cyst walls (E. C. Wootton, unpublished), suggesting that Himasthla sp. secretory products were not responsible for the lack of encapsulation towards some cysts. Why only one third of cysts induce a host response is currently unknown.
The nature of the invertebrate encapsulation response is highly
species-specific, but the basic mechanisms, involving non-specific
physicochemical factors such as electrostatic forces, are potentially common
to all encapsulation reactions. It is the modification of these basic
mechanisms, by highly specific biochemical interactions, which produce unique
responses to individual host–parasite associations. These biochemical
interactions are determined by many factors including parasite species,
viability and stage of development as well as the susceptibility of the host
and the particular host tissue invaded (e.g.
Cheng et al., 1966;
Lie et al., 1987;
Ataev et al., 1999;
Laruelle et al., 2002). The
gastropod mollusc, Biomphalaria glabrata, for example, exhibits a
variety of encapsulation responses towards invading trematodes, including
haemocytic infiltration of rounded haemocytes, type I capsules involving
flattened cells, type II capsules involving polygonal cells, and amoebofibrous
capsules (Lie et al., 1987).
In addition, five distinct encapsulation types have been described in bivalve
molluscs in response to such parasites
(Cheng and Rifkin, 1970),
including aggregation, as observed in the present work. It is justified,
therefore, to designate `encapsulation' to describe the haemocytic responses
exhibited by C. edule during the present study.
All our experiments provided strong evidence for a role for surface charge and non-specific electrostatic forces in the encapsulation response of C. edule. Investigations, both in vivo and in vitro, into rate of encapsulation revealed that all positively charged targets elicited the strongest and most rapid response, regardless of their functional group, whilst negatively charged and neutral targets mediated a less vigorous response. This illustrates that haemocytes are responding to surface charge and not surface molecular patterns and this may be partly due to the beads not possessing common PAMPs, such as LPS, peptidoglycan or ß-1,3 glycan.
Further experiments, in vitro, revealed that live and dead cells (both with and without plasma) associated with, or encapsulated, positively charged targets. This contrasts with negatively charged targets, which were only encapsulated by living haemocytes in the presence of plasma. These observations, combined with the enhancement of cell attachment through changing the bead surface charge from negative to positive, and the converse change in bead surface charge lowering the haemocytic response, strongly imply that non-specific electrostatic forces are influencing bead–haemocyte interactions. As C. edule haemocytes were shown to be negatively charged, electrostatic attractions between haemocytes and positively charged beads are entirely feasible.
In our experiments, heparin, despite its anticoagulant properties, was used
to reverse the surface charge of positive DEAE Sepharose beads. It was
considered an appropriate compound as its binding forces are largely
electrostatic (Lindahl, 1997).
In addition, the prophenoloxidase (PpO) cascade in C. edule is
minimal as cockles do not exhibit melanization reactions (E. C. Wootton,
unpublished), nor do the haemocytes contain phenoloxidase
(Wootton et al., 2003). Thus,
the interaction of heparin with immunological serine proteases to activate a
prophenoloxidase system is unlikely. To confirm that heparin was not
interfering with such proteins, two additional compounds,
poly-L-glutamate and poly-L-asparate, were also used to
reverse the surface charge of DEAE Sepharose beads, and these beads produced
very similar encapsulation responses to those bound with heparin.
In other invertebrates, particularly insects, positively charged targets
commonly elicit strong encapsulation responses
(Walter and Williams, 1967;
Vinson, 1974;
Dunphy and Nolan, 1980;
Lackie, 1983;
Ratner and Vinson, 1983), and
non-specific electrostatic forces have been considered an influential factor
(e.g. Walters and Williams, 1967; Vinson,
1974; Lackie,
1983; Wiesner,
1992). It has been suggested that humoral recognition molecules
bind more readily to positively charged targets
(Vinson, 1974;
Wiesner, 1992;
Strand and Pech, 1995),
mediating `non-self' recognition and immune defence reactions. It has also
been proposed that negatively charged targets adsorb humoral components,
become disguised as `self', and thus reduce host immune responses
(Walter and Williams, 1967;
Paskewitz and Riehle, 1994).
In addition, the stability of recognition molecules in the vertebrate
complement pathway increases on contact with surfaces of a particular charge
(Toufik et al., 1995), and
this has been suggested to occur in insect immunity too
(Lackie, 1988). As of yet,
these hypotheses explaining enhanced encapsulation of positively charged
targets by invertebrate haemocytes have not been fully tested
Thus, non-specific electrostatic forces are probably only one of many
factors involved in cell attachment during immune defence reactions. Opsonins
and pattern recognition receptors, for example, will undoubtedly play an
important role. In this respect, our study, like many others (e.g.
Davies et al., 1988;
Paskewitz and Riehle, 1998;
Lavine and Strand, 2001),
revealed a strong involvement of humoral components. This is highlighted by
the encapsulation of negatively charged targets exclusively by living
haemocytes in the presence of plasma. The specificity of this reaction was
confirmed by the lack of encapsulation of negatively charged beads in the
presence of the plasma substitute, FCS, and by the opsonization of beads only
by pre-incubation in C. edule plasma. In the present study, humoral
opsonisation appeared to reduce the electrostatic repulsion between the
negatively charged haemocytes and negatively charged targets, possibly through
electrostatic interactions, thus allowing cells and beads to interact. Humoral
factors, therefore, mediate `non-self' recognition processes in C.
edule and allow for encapsulation of targets with different surface
charges. This is important for effective immune defence, as many parasites and
pathogens, including Himasthla sp. cysts, carry a negative surface
charge, which is thought to be an evasive strategy to avoid host immune
responses (Crocker and Varki,
2001). Dead haemocytes in the presence of plasma, however, did not
attach to negatively charged targets, and this highlights that humoral
opsonization, and subsequent encapsulation, involves active cell-to-cell
communication.
Humoral opsonisation, however, does not completely override the non-specific electrostatic forces. Our study shows that the two factors act simultaneously in influencing cell attachment. For example, positively charged targets are more vigorously encapsulated than negatively charged or uncharged targets in the presence of plasma. The electrostatic attraction between positively charged targets and negatively charged haemocytes is an additional influence to humoral opsonisation, and thus enhances the response. In addition, although dead cells attached to positively charged beads in the presence of plasma, the response was weaker than that of dead cells in the absence of plasma. This reveals that humoral factors were interfering with, but not completely masking, the electrostatic attraction between cells and beads. As the haemocytes were dead, cell attachment must be due to non-specific electrostatic forces, because, as suggested previously, encapsulation requires active cell-to-cell communication.
Although previous studies in insects have often postulated that surface
charge is an important influencing factor of haemocytic encapsulation in
invertebrates (e.g. Walters and Williams, 1967;
Vinson, 1974;
Lackie, 1983;
Wiesner, 1992;
Paskewitz and Riehle, 1994),
detailed investigations into proving this hypothesis are very limited. Lackie
(Lackie, 1981) proposed that
immune recognition in insects is based on a `two-tiered' system, in which
electrostatic forces have an additive or synergistic effect on the highly
specific biochemical `non-self' recognition processes. Our results support the
`two-tiered' hypothesis by highlighting the importance of electrostatic
interactions in cellular communication, and by providing substantial evidence
for an interactive and influential role for both non-specific electrostatic
forces and humoral factors in haemocyte attachment and encapsulation.
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Acknowledgments |
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Footnotes |
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Present address: School of Life Sciences, Heriot-Watt University, Edinburgh
EH14 4AS, UK 
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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