Carbohydrates that mimic schistosome surface coat components affect ERK and PKC signalling in Lymnaea stagnalis haemocytes

International Journal for Parasitology 35 (2005) 293–302 www.parasitology-online.com

Carbohydrates that mimic schistosome surface coat components affect ERK and PKC signalling in Lymnaea stagnalis haemocytes Louise D. Plows, Richard T. Cook, Angela J. Davies, Anthony J. Walker* School of Life Sciences, Kingston University, Penrhyn Road, Kingston-Upon-Thames, Surrey KT1 2EE, UK Received 16 September 2004; received in revised form 10 November 2004; accepted 11 November 2004

Abstract Molluscs are intermediate hosts for helminth parasites such as Schistosoma spp. that possess an immunogenic surface coat of high carbohydrate content, with fucose as the predominant saccharide. More than a decade ago, it was postulated that such components could block receptors on snail haemocytes thus preventing recognition of intra-molluscan schistosome stages. Although more recent studies have shown that carbohydrates can suppress processes such as phagocytosis by haemocytes, interference of the haemocyte cell signalling pathways that regulate immunity by saccharides has not yet been investigated. We have recently reported the presence of extracellular-signal regulated kinase and protein kinase C in Lymnaea stagnalis haemocytes. Here we show that extracellular-signal regulated kinase and protein kinase C activities are down-regulated when haemocytes are exposed to albumin-linked fucose and galactose in the absence of haemolymph. Moreover, we demonstrate that phagocytosis is reduced under these conditions. Interestingly, in the presence of haemolymph, only protein kinase C activity is down-regulated and only galactose suppresses phagocytosis, implying a role for serum factors in the preservation of haemocyte function following exposure. We therefore propose that the establishment of a compatible relationship between a schistosome and its snail host is at least in part due to down-regulation of cell signalling events in haemocytes. q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Phagocytosis; MAPK; Glycoconjugate; PKC; Haemolymph; Molluscan defence

1. Introduction Schistosome parasites use gastropod molluscs as their intermediate hosts, entering the snail as miracidia, then subsequently transforming into mother and daughter sporocysts before emerging as free-swimming cercariae in the freshwater environment. The effects of parasitism on the physiology of the snail host have been studied widely in Biomphalaria glabrata and Lymnaea stagnalis, host to the human schistosome Schistosoma mansoni, and the avian schistosome Trichobilharzia ocellata, respectively. Such physiological changes in the snail host include alterations to endocrine function, metabolism and immune regulation (De Jong Brink, 1995; De Jong Brink et al., 1999; Amen et al., 1992; Boissier et al., 2003). * Corresponding author. Tel.: C44 20 8547 2000x62466; fax: C44 20 8547 7562. E-mail address: [email protected] (A.J. Walker).

Molluscs have a potent innate defence system that consists of both cellular and humoral defence mechanisms. The humoral components include bacteriostatic, bactericidal, cytolytic and antiviral factors as well as proteinase inhibitors. In addition, lectins (carbohydrate-binding proteins) are thought to play a major role in the humoral defence system acting as key recognition factors for nonself. Haemocytes, mobile phagocytic cells that functionally resemble mammalian macrophages, carry out the cellular arm of immunity. These immune cells are responsible for phagocytosis of invading bacteria or encapsulation of larger foreign bodies such as parasites. Lectins are synthesised by both haemocytes and the albumin gland and can be found reversibly bound to the haemocyte surface or free in the haemolymph. These lectins are capable of binding to carbohydrate moieties on the surface of parasites thus enabling recognition by haemocytes and subsequent activation of the snail immune response (reviewed by Horak and van der Knaap, 1997).

0020-7519/$30.00 q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2004.11.012

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In the early 1990s, a number of studies focused on the effects of T. ocellata infection on L. stagnalis immunity (Amen et al., 1992; Amen et al., 1991a,b; Nunez et al., 1994). During this period it was also postulated that carbohydrates present on the schistosome surface coat could block receptors on snail haemocytes thus preventing recognition of the intramolluscan parasite stage (Hamburger et al., 1992). The helminth surface coat comprises predominantly fucose and galactose, with galactosamine and glucosamine present in lower quantities (Khoo et al., 1995; Cummings and Nyame, 1999). Most studies have focused on the surface coat of S. mansoni, but the biosynthesis of structural elements in T. ocellata cercariae is comparable to that described for S. mansoni, suggesting similarities in the surface coat saccharides between the two parasite species (Hokke et al., 1998). Although studies have shown that carbohydrate moieties known to be expressed on the schistosome surface can modulate haemocyte immune responses, such as the production of reactive oxygen species and phagocytosis (Hahn et al., 2000; Hora´k et al., 1998), interference of haemocyte signalling pathways by such compounds has never been reported. We have recently demonstrated the presence of the protein kinase C (PKC) (Walker and Plows, 2003) and extracellular-signal regulated kinase (ERK) signalling pathways (Plows et al., 2004) in L. stagnalis haemocytes, and have shown that these pathways regulate phagocytosis of Escherichia coli ‘bioparticles’ (Plows et al., 2004). The ERK pathway consists of three components in a hierarchical phosphorylation cascade, Raf, Mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) and ERK. MEK is activated by the protein kinase Raf, which is itself phosphorylated in either a Ras-dependent or Ras-independent manner (Bu¨scher et al., 1995; Chen et al., 1996). PKC can activate the ERK pathway in both mammalian macrophages (Monick et al., 2000) and molluscan haemocytes (Humphries et al., 2001). Phosphorylated ERK can then target cytosolic components such as cytoskeletal elements (Reszka et al., 1995), or can translocate to the nucleus where it activates various transcription factors including Elk-1 (Aplin et al., 2001). Here we show that the activities of the L. stagnalis haemocyte ERK and PKC signalling cascades are altered following exposure to albumin-linked fucose and galactose. This is the first report to demonstrate that carbohydrate moieties known to be present on the schistosome surface can interfere with haemocyte cell signalling events. In addition, we show that the phagocytic activity of haemocytes is suppressed when they are exposed to the albuminlinked sugars in the absence of haemolymph and that this effect is at least in part due to a reduction in kinase activity. Interestingly, the presence of haemolymph seems to help preserve ERK activity and phagocytosis in haemocytes. These findings have implications for host immunity to schistosomes and we hope that our work will stimulate further research in this area.

2. Materials and methods 2.1. Snails Laboratory cultures of L. stagnalis were reared from eggs that were produced by adult snails purchased from Blades Biologicals (Edenbridge, UK). Juvenile snails were kept at room temperature until they reached a shell length of 20–30 mm, they were then transferred to an incubator and kept under a 12 h light-dark cycle at 20 8C. All tanks contained continuously aerated water, which had been filtered through a Brimak/carbon filtration unit (Silverline Ltd, Winkleigh, UK). Water was changed weekly and snails were fed fresh lettuce ad libitum. 2.2. Haemolymph extraction and haemocyte treatments Adult L. stagnalis were washed with distilled water and the haemolymph was extracted from the snails by head-foot retraction (Sminia, 1972). Haemolymph from 15 to 20 snails was collected, pooled, and kept on ice in sterile snail saline (SSS: 3 mM Hepes, 3.7 mM NaOH, 36 mM NaCl, 2 mM KCl, 2 mM MgCl2, 4 mM CaCl2, pH 7.8, sterilised through a 0.22 mm disposable filter) (Sminia, 1972; 1 part SSS:2 parts haemolymph). This diluted haemolymph contained approximately 3!106 viable haemocytes per ml. Haemocyte monolayers were then prepared by allowing cells to adhere to individual wells (200 ml diluted haemolymph per well) of a 24-well culture plate (Nunc) for 30 min at room temperature. Almost all of the haemocytes adhered during this time period since few cells were observed in the SSS removed at the first wash step (Section 2.3). 2.3. SDS-PAGE and Western analysis In preparation for Western analysis, haemocyte monolayers were washed three times with SSS and were allowed to equilibrate for a further 15 min at room temperature. This equilibration period was deemed sufficient since longer periods of equilibration (up to 80 min) do not affect basal levels of ERK phosphorylation (Plows et al., 2004); a similar response is observed with PKC (unpublished data). SSS was then removed and bovine serum albumin (BSA) conjugated galactose (A 5908, Sigma, Poole, UK) or fucose (A 6033, Sigma) were added (0–800 nM in SSS), either individually or in combination (1:1; 0–800 nM of each in SSS). These carbohydrate conjugates comprised 15–25 sugar molecules per BSA molecule and concentrations used represent the final molarities of the conjugated BSA in the assays. In initial experiments, control haemocytes were exposed to BSA at the appropriate dose (0–800 nM); however, control phosphorylation levels were similar regardless of BSA dose. Consequently, in subsequent experiments, control haemocytes were exposed to 800 nM BSA or to SSS. For experiments conducted in the presence of haemolymph, cells were not washed and sugars were

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diluted in the haemolymph serum. After 1 h exposure, the cells were lysed with boiling SDS-PAGE sample buffer. This period of incubation was determined from preliminary experiments in which four time periods over 90 min were tested (0, 15, 30, 60, 90 min). Although the effects on the phosphorylation levels observed were greatest after 60 min, there were no large differences between 15 and 90 min. Samples were then either processed immediately for SDSPAGE and Western blotting or were stored at K20 8C. Samples containing equal amounts of protein were loaded onto a discontinuous SDS-PAGE slab gel, which contained 10% acrylamide in the resolving gel. After electrophoresis, proteins were electro-blotted onto Hybond nitrocellulose membrane (0.45 mm; Amersham Biosciences, Amersham, UK), using a semi-dry transfer unit and homogeneous transfer was confirmed by staining with Ponceau S. Membranes were then blocked for 1 h at room temperature with 5% (w/v) non-fat dried milk in Tris-buffered saline containing 0.1% (v/v) Tween-20 (TTBS). Membranes were incubated overnight at 4 8C with agitation in anti-phosphop44/42 MAPK (1:1000; New England Biolabs, Beverly, USA), anti-phospho PKC (pan) (1:1000; New England Biolabs) or anti-actin (1:5000; Sigma) primary antibodies. Immunoreactive proteins were then visualised, after exposure to horseradish peroxidase-conjugated secondary antibody (Bio-Rad, Hemel Hempstead, UK) for 1 h at 25 8C, with the Opti-4CN detection kit (Bio-Rad). No signal was detected when membranes were incubated in secondary antibody alone. The intensity of the immunoreactive signal on individual blots was analysed using Kodak 1D Image analysis software. Values were calculated as a net difference in phosphorylation levels with treatment, compared to the control value for each replicate; hence, each control value was assigned a standardised value of 1. Five replicate experiments were carried out for the analysis of each pathway (PKC or MAPK), and each replicate was performed on a different day with a different batch of snails.

the anti-phospho p44/42 MAPK antibody (1:150 in blocking buffer; 3 h), followed by a FITC-conjugated goat-anti-rabbit secondary antibody (1:150 in blocking buffer; 45 min; Sigma) and rhodamine phalloidin (0.1 mg/ml; 40 min; Sigma). All steps were performed at room temperature and incubations were done in humidified chambers. Finally, coverslips were mounted onto slides with Vectashield (Vector Laboratories, USA) and were observed with a Zeiss Axiophot 20 photomicroscope using a triple filter; excitation wavelengths were 410, 505 and 585 nm (with beamsplitters: 395, 485 and 560 nm; and barriers: 460, 530 and 610 nm, respectively). Images were digitally captured using a Nikon DN100 camera linked to the Nikon Eclipse Net image analysis package.

2.4. Immunocytochemistry

Where appropriate, results were analysed with one-way analysis of variance (ANOVA) and post-hoc multiple comparison tests (Tukey), using the statistical software package SPSS. Two-way ANOVA was used to compare results obtained from phagocytosis assays with and without haemolymph. Independent t-tests were also done between experiments with and without haemolymph at specific concentrations.

Haemocytes were allowed to adhere to coverslips for 30 min at room temperature before being washed with SSS three times. After equilibration in SSS for 15 min, SSS was removed and haemocytes were exposed to BSA-conjugatedfucose (800 nM in SSS) and -galactose (800 nM in SSS), either separately or in combination (1:1, 800 nM of each in SSS) for 1 h. Control haemocytes were exposed to either BSA (800 nM) or SSS. For experiments in which cells were incubated in sugars with haemolymph, albumin-linked sugars were diluted in haemolymph and cells were not washed after the binding period. Haemocytes were subsequently fixed and permeabilised with fixing buffer (3.7% (v/v) formaldehyde (Sigma), 0.18% (v/v) Triton X-100 (Sigma) in phosphate buffered saline (PBS) (Oxoid)) for 12 min, and coverslips blocked with BSA (Sigma; 1% (w/v) in PBS) for a further 12 min. Cells were then incubated with

2.5. Phagocytosis assay Haemocyte monolayers were prepared as previously described in individual wells of a 96-well culture plate (Nunc, 100 ml diluted haemolymph per well) and washed three times with SSS. For assays with haemolymph serum, cells were not washed and haemolymph remained in the wells throughout the experiment. Haemocytes were then pre-incubated with BSA-conjugated-galactose (0–800 nM in SSS) or -fucose (0–800 nM in SSS), either separately or in combination (1:1, 0–800 nM of each in SSS), for 30 min prior to the addition of FITC conjugated ‘bioparticles’ (6!106 per well; Sigma). After the haemocytes had been challenged with ‘bioparticles’ for 1 h at room temperature in a dark chamber, the ‘bioparticles’ were removed and 2% (w/v) trypan blue (Sigma) was added to the wells for 2 min to quench extracellular fluorescence. Intracellular fluorescence was then quantified using a Fluorstar Optima microplate spectrofluorometer (BMG Labtechnologies, Aylesbury, UK). 2.6. Statistical analysis

3. Results 3.1. Albumin-linked sugars alter ERK and PKC phosphorylation To determine whether or not haemolymph components could alter the responses of haemocytes to sugars, studies were carried out in the presence or absence of haemolymph.

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Fig. 1. Extracellular-signal regulated kinase pathway activity is modulated by bovine serum albumin-conjugated sugars. Haemocytes were exposed without haemolymph (A, upper panel) and with haemolymph (A, middle panel) to albumin-linked galactose, fucose and combined sugars at 200, 400 and 800 nM; control haemocytes were exposed to bovine serum albumin alone. Actin was probed as a protein control (A; lower panel). Densitometric analysis was performed on blots and the relative change in phosphorylation values with reference to the control value of 1.00 (see Section 2) shown as the dotted line, were calculated; mean values from five independent experiments are shown (GSEM). Shaded bars and white bars represent values from experiments with and without haemolymph, respectively. (*P%0.05; **P%0.01 when compared to control values).

Without haemolymph, exposure of haemocytes to either BSA-galactose or BSA-fucose resulted in reduced levels of ERK phosphorylation, compared to BSA controls (Fig. 1A, upper panel). Levels of ERK phosphorylation under basal (control) conditions were similar regardless of whether haemocytes were exposed to BSA or SSS (data not shown). The effect of sugar exposure on ERK phosphorylation was most prominent when haemocytes were exposed to 400 nM BSA-galactose or 800 nM BSA-fucose, since at these concentrations ERK phosphorylation was significantly reduced by 54 and 50%, respectively (P%0.05, Fig. 1B). Moreover, the inhibitory effect of albumin-linked fucose on ERK phosphorylation appeared to be dose-dependent. The combined use of albumin-linked sugars also resulted in a dose-responsive decline in ERK phosphorylation (Fig. 1A), with 800 nM of BSA-galactose/BSA-fucose reducing phosphorylation by 67% (P%0.01; Fig. 1B). PKC phosphorylation levels were also reduced when haemocytes were

exposed to sugars in the absence of haemolymph (Fig. 2). The effect of BSA-galactose was dose-responsive with phosphorylation levels being significantly reduced by 56 and 73% at 400 and 800 nM, respectively, when compared with BSA controls (P%0.01 and P%0.001; Fig. 2B). As observed for ERK, levels of PKC phosphorylation under basal (control) conditions were similar regardless of whether haemocytes were exposed to BSA or SSS (data not shown). Phosphorylation of haemocyte PKC was also reduced in the presence of BSA-fucose, but at the higher doses, the inhibitory effects were less than those observed for galactose (Fig. 2B). Interestingly, the combined use of BSA-galactose and BSA-fucose gave highly significant reductions in PKC phosphorylation at all three concentrations (Fig. 2A upper panel), with individual sugar concentrations of 200, 400, and 800 nM suppressing phosphorylation levels to 36, 24 and 8% of the control, respectively (P%0.001; Fig. 2B).

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Fig. 2. Protein kinase C pathway activity is modulated by bovine serum albumin-conjugated sugars. Haemocytes were exposed without haemolymph (A, upper panel) and with haemolymph (A, middle panel) to albumin-linked galactose, fucose and combined sugars at 200, 400 and 800 nM; control haemocytes were exposed to bovine serum albumin alone. Actin was probed as a protein control (A; lower panel). Densitometric analysis was performed on blots and the relative change in phosphorylation values with reference to the control value of 1.00 (see Section 2) shown as the dotted line, were calculated; mean values from five independent experiments are shown (GSEM). Shaded bars and white bars represent values from experiments with and without haemolymph, respectively. (*P%0.05; **P%0.01, ***P%0.001 when compared to control values).

In contrast to when haemolymph was absent, in the presence of haemolymph, haemocyte ERK phosphorylation was not significantly altered (compared to BSA controls), when sugars were either delivered independently or in combination (Fig. 1A, middle panel). Image analysis of individual blots revealed that fucose had in some cases increased ERK phosphorylation, although no overall significant increase was found (Fig. 1B). In contrast to ERK phosphorylation, PKC phosphorylation levels were reduced in the presence of haemolymph when BSA-sugars were delivered either separately or in combination (Fig. 2A, middle panel). Whereas BSA-galactose only caused a significant reduction (45%) in PKC phosphorylation at 800 nM (P%0.001; Fig. 2B), BSA-fucose resulted in significant reductions of PKC phosphorylation at all concentrations (Fig. 2A, middle panel). Exposure to albumin-linked fucose at 200, 400 and 800 nM resulted in

PKC phosphorylation levels being reduced to 68% (P%0.05), 65% (P%0.05) and 36% (P%0.001) of control levels, respectively (Fig. 2B). When used in combination, BSA-galactose and BSA-fucose inhibited PKC phosphorylation more than ERK phosphorylation, with all concentrations of sugars mediating significant effects (P%0.001). 3.2. Exposure of haemocytes to albumin-linked galactose changes localisation of active ERK Immunocytochemistry revealed that phosphorylated ERK isozymes are cytoplasmic in cells exposed to BSA alone (control; Fig. 3A). The intensity and distribution of the phosphorylated ERK signal in haemocytes exposed to SSS did not differ from that seen with BSA (data not shown). When haemocytes were exposed to BSA-galactose (800 nM), both in the presence and absence of haemolymph,

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Treatment of cells with BSA-conjugated sugars also appeared to increase the degree of haemocyte spreading (Fig. 3A–C). 3.3. Haemocyte phagocytic activity is affected by albumin-linked sugars

Fig. 3. Immunocytochemistry reveals the location of active extracellularsignal regulated kinase following sugar exposure. Haemocytes were exposed to bovine serum albumin (control; A), 800 nM galactose without haemolymph (B), and 800 nM galactose with haemolymph (C). Rhodamine phalloidin was used to stain filamentous actin (red), and anti-phosphop44/42 mitogen-activated protein kinase antibody was used to stain active extracellular-signal regulated kinase (green). Results are representative of three independent experiments.

phosphorylated ERK isozymes were found to be located at the cell periphery (Fig. 3B and C). When cells were exposed to BSA-fucose, the distribution of phosphorylated ERK was broadly similar to that seen with BSA-galactose.

Haemocytes exposed to BSA-fucose in the absence of haemolymph displayed significantly reduced phagocytic activity when compared to BSA controls (P%0.001), with 800 nM BSA-fucose resulting in the lowest level of ‘bioparticle’ internalisation (9% of control levels; Fig. 4A). In contrast, in the presence of haemolymph, the highest concentration of BSA-fucose (800 nM), significantly increased haemocyte phagocytic activity by 77% (P%0.05; Fig. 4A). As expected, two-way analysis of variance revealed that there was a significant effect of haemolymph on the phagocytic activities following sugar exposure (P%0.001). In contrast to the effects of BSA-fucose, phagocytosis was reduced following exposure to BSA-galactose irrespective of whether or not haemolymph was present (Fig. 4B). In the absence of haemolymph, phagocytosis was significantly reduced to 12% of BSA control levels when haemocytes were exposed to 800 nM BSA-galactose, with lower concentrations of BSA-galactose also mediating significant effects (P%0.001). When haemolymph was present, the highest dose of galactose (800 nM) reduced haemocyte phagocytic activity to 2% of control levels (P%0.001). In addition, independent t-tests revealed significant differences between levels of phagocytosis with and without haemolymph following galactose exposure (P%0.01). One-way analysis of variance revealed that phagocytosis was also significantly reduced by all concentrations of the combined sugars, in the presence or absence of haemolymph, when compared to BSA controls (P%0.001; Fig. 4C). The effect of combined sugar exposure on phagocytic activity was however reduced when haemolymph was present, such that haemolymph preserved the phagocytic activity to approximately four to five times that seen in the absence of haemolymph at all sugar concentrations (Fig. 4C). Furthermore, two-way analysis of variance revealed significant differences between phagocytic activity in the presence and absence of haemolymph following sugar exposure (P%0.001).

4. Discussion Since molluscs are important intermediate hosts for schistosome parasites that possess carbohydrate-rich surface coats, it is surprising that the effects of such carbohydrates on mollusc immune cells have been largely overlooked at a molecular level. In this study we have demonstrated that fucose and galactose, sugars commonly found on surface coats of schistosome larval stages, are capable of causing

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Fig. 4. Lymnaea stagnalis haemocyte phagocytic activity is affected by bovine serum albumin-sugars. Levels of phagocytic activity of haemocytes exposed to bovine serum albumin-fucose (A), bovine serum albumin-galactose (B), and combined sugars (C) are shown in the presence (shaded bars) and absence of haemolymph (white bars); control haemocytes were exposed to bovine serum albumin alone. Values shown are means (GSEM) (*P%0.05 and ***P%0.001, compared with control group; nZ12).

down-regulation of ERK and PKC signalling in L. stagnalis haemocytes. These sugars also affect the phagocytic activity of haemocytes, an effect that is at least in part due to the modulation of ERK and PKC signalling pathways. The differences in ERK signalling and phagocytosis observed when haemolymph was present compared to when it was absent, suggest that serum components play an important role in the recognition of fucose and galactose monosaccharides.

The anti-phospho p44/42 MAPK and anti-phospho PKC (pan) antibodies recognise the active forms of their respective kinases and as such have been used in numerous studies to detect pathway activation. We have recently validated these antibodies for use in L. stagnalis and have demonstrated that the phosphorylated immunoreactive proteins detected in haemocytes are indeed L. stagnalis ERK and PKC, respectively (Walker and Plows, 2003;

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Plows et al., 2004). In the present study, in the absence of haemolymph the activity of both ERK and PKC was reduced by BSA-fucose and BSA-galactose, especially when sugars were used in conjunction. We have previously reported that ERK and PKC activity is required for efficient phagocytosis by L. stagnalis haemocytes (Plows et al., 2004), and we report here that, as expected, in the absence of haemolymph, phagocytic activity generally reflected ERK and PKC activity. Whilst other researchers have reported the effects of sugars, such as arabinose and fructose, on haemocyte phagocytic activity (Hora´k et al., 1998), the present study is the first to demonstrate that key intracellular signalling pathways can be modulated by such sugars leading to downstream effects on immune function. Although in this study, haemocytes were exposed to ‘bioparticles’ in the presence of sugars, we cannot rule out the possibility that the sugars may be binding to the ‘bioparticles’ as well as the haemocyte surfaces. However in the absence of ‘bioparticles’ the effects of sugars on the activity of ERK and PKC pathways demonstrate a direct effect of sugars on haemocytes. With haemolymph present, the contrasting results between PKC and ERK activation following exposure of haemocytes to BSA-galactose and BSA-fucose were surprising. Activity of the ERK pathway was not significantly reduced, and in some cases, fucose appeared to upregulate ERK activation, although not significantly. In contrast, PKC activity appeared to be inhibited in a doseresponsive manner in the presence of haemolymph, particularly when the two sugars were combined. Taken together these results suggest that within snails haemocyte signalling mechanisms can be differentially regulated by carbohydrate exposure. As phosphorylation of PKC appears to be inhibited in the presence of the sugars, but ERK remains active, PKC does not seem to be a key mediator of ERK-dependent responses in haemocytes following exposure to these carbohydrates. ERK can, however, be activated by numerous other signalling components including Protein Kinase A (PKA) (Cancedda et al., 2003; Schmitt and Stork, 2002; Miggin and Kinsella, 2002), Phosphatidylinositol-3-kinase (PI-3-K) (Versteeg et al., 2000) and integrin binding which may involve Focal Adhesion Kinase (FAK) (Irigoyen and Nagamine, 1999; Yu and Basson, 2000; Barberis et al., 2000; Yujiri et al., 2003). The phagocytic responses of haemocytes in the presence of haemolymph differed following BSA-galactose and BSA-fucose exposure. Albumin-linked galactose inhibited phagocytosis dramatically in a dose-responsive manner, whereas fucose significantly increased haemocyte phagocytic activity by 77%. In both cases, there were no significant changes in ERK phosphorylation levels. The differing effects of BSA-galactose and BSA-fucose on phagocytosis could be due to haemolymph components and fucose acting in concert as an opsonin, thus up-regulating the phagocytosis of ‘bioparticles’. This is plausible since molluscan serum lectins are known to be able to act as

opsonins, as discussed by Horak and Van der Knaap (1997). Clearly, further research into L. stagnalis haemolymph lectins needs to be undertaken to gain further insight into the enhancing effect of fucose on phagocytosis. Given that galactose suppresses phagocytosis considerably and that we have previously shown that ERK activity is required for phagocytosis (Plows et al., 2004) we were surprised that galactose did not significantly reduce ERK activation. Levy et al. (2003) have recently reported that galectins (galactose-specific lectins) can bind to integrins on the membrane of Chinese Hamster Ovary (CHO) cells. Integrins are known to be part of the cellular machinery used for phagocytosis in insect haemocytes (Foukas et al., 1998), and galactose specific lectins have been isolated from many invertebrate species (Pace et al., 2002; Hirabayashi et al., 1998; Mann et al., 2000; Chiou et al., 2000). In addition, we have shown that the integrin blocking peptide, RGDS, is capable of reducing phagocytosis in a dose dependent manner in L. stagnalis haemocytes (unpublished data). Integrin activation has been linked to increased ERK activity in a number of cell types including thyroid TAD-2 cells, Hep-3B cells (Illario et al., 2003), neuronal, gonadal and Swiss-3T3 cells (Barberis et al., 2000). Therefore, the low levels of phagocytosis and high levels of active ERK in our system may be due to galectins binding to galactose and subsequent engagement of integrins. Immunocytochemistry revealed that following exposure of haemocytes to BSA-galactose, in the presence or absence of haemolymph, phosphorylated ERK was located mainly at the cell periphery of haemocytes, but was also diffuse throughout the cytoplasm. This finding contrasts our previous work with E. coli lipopolysaccharide (LPS) in which we found that LPS challenge resulted in a perinuclear distribution of active ERK and very little at the periphery of the cell (Plows et al., 2004). The presence of active ERK at the cell periphery following carbohydrate exposure further suggests integrin engagement/activation or ERK binding to focal adhesion sites associated with integrins. These sites are comprised of enzymes such as Focal adhesion Kinase (FAK), an activator of Raf in mammalian fibroblasts (Yujiri et al., 2003). Clearly, that integrins may be involved in carbohydrate recognition and downstream signalling in molluscan haemocytes needs further investigation. To our knowledge, this is the first report demonstrating altered PKC and ERK signalling in molluscan immune cells in response to challenge with albumin-linked sugars. BSAgalactose and BSA-fucose were chosen since they have been shown to modulate molluscan immune responses (Hahn et al., 2000), and because galactose and fucose are major components of the schistosome antigens LacdiNac (LDN) and fucosylated LacdiNac present on intramolluscan stages of the parasite (Nyame et al., 2002). Results obtained in the presence of haemolymph using a combination of sugars may be more representative of a helminth infection, since haemocytes will be exposed to more than one sugar, and snail serum components will also be present.

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Interestingly, with haemolymph and both sugars combined, phagocytosis was significantly reduced suggesting a possible mechanism of host immune evasion by schistosome larvae. Although phagocytosis and encapsulation are different biological responses, the initial recognition events and their effects on downstream signalling pathways are likely to be similar. Our results suggest that parasitemediated down-regulation of signalling events to facilitate immune evasion could be a strategy employed by extracellular parasites. It is well established that intracellular parasites interfere with host immune function by altering signalling pathway activities and recently Leishmania has been shown to secrete phosphatases to switch off ERK signalling in macrophages, thus increasing its chance of survival (Martiny et al., 1999). Clearly, further research is needed to fully understand the mechanisms of carbohydrate recognition by haemocytes. A future plan is to challenge haemocytes with sporocyst surface coats to see if they affect PKC and ERK signalling and modulate behavioural responses in a similar way to BSA-fucose and BSA-galactose. Further characterisation of L. stagnalis serum and cell-surface lectins is also required to fully understand the role of humoral arm of immunity in the molluscan immune response. Such research would help elucidate how lectin-carbohydrate binding to the haemocyte surface results in the modulation of intracellular signalling pathways.

Acknowledgements This study was part funded by a grant from the Royal Society awarded to AJW.

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