- Email: [email protected]
Phagocytosis by Lymnaea stagnalis haemocytes: A potential role for phosphatidylinositol 3-kinase but not protein kinase A
Journal of Invertebrate Pathology 91 (2006) 74–77 www.elsevier.com/locate/yjipa
Research note
Phagocytosis by Lymnaea stagnalis haemocytes: A potential role for phosphatidylinositol 3-kinase but not protein kinase A Louise D. Plows 1, 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 2005; accepted 26 October 2005
Abstract The molecular events that regulate phagocytosis, an important innate immune response, in invertebrate defence cells (haemocytes) are poorly understood. Lymnaea stagnalis haemocytes were used as a model to elucidate the role of cell signalling pathways in phagocytosis by molluscan defence cells. The phosphatidylinositol 3-kinase (PI3-K) inhibitor, LY294002, signiWcantly impaired haemocyte phagocytic activity in a dose-responsive manner with 10 M LY294002 reducing internalization of Xuorescent-conjugated Escherichia coli by 62% (P 6 0.001). In contrast, the protein kinase A (PKA) inhibitor KT5720 was without eVect. Therefore, PI3-K, but not PKA, appears to control phagocytosis by haemocytes in these gastropod molluscs. 2005 Elsevier Inc. All rights reserved. Keywords: Lymnaea stagnalis; Snail; Invertebrate defence; E. coli; Protein kinase A; Phosphatidylinositol 3-kinase
Mobile phagocytic cells called haemocytes play a major part in limiting infection in invertebrates. To date our knowledge of the molecular mechanisms that regulate the defence behaviour of these macrophage-like cells remains fragmentary. This is particularly true for the haemocytes of molluscs which facilitate the removal or sequestration of a range of infectious organisms including bacteria and incompatible parasites (Van der Knaap et al., 1993). Recently, we have identiWed two key signalling enzymes, protein kinase C (PKC) and extracellular signal-regulated kinase (ERK), in freshly collected (primary) haemocytes of the model snail Lymnaea stagnalis (Plows et al., 2004; Walker and Plows, 2003). Our work on these signalling proteins revealed that their activities are modulated by bacterial products and that both pathways are key regulators of the phagocytic response. Other workers have shown that these pathways mediate the spreading behaviour of a haemocyte-like, embryonic cell line (Bge) from the snail Biom*
Corresponding author. Fax: +44 0 20 8547 7562. E-mail address: [email protected] (A.J. Walker). 1 Present address: The Kennedy Institute of Rheumatology, Faculty of Medicine, Imperial College, ARC Building, 1 Aspenlea Road, London W6 8LH, UK. 0022-2011/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2005.10.011
phalaria glabrata, intermediate host to the human schistosome, Schistosoma mansoni (Humphries and Yoshino, 2001). By inhibition assay, primary haemocytes from B. glabrata have also been shown to rely on PKC activity for phagocytosis (Humphries and Yoshino, 2003). A number of other signalling enzymes either positively or negatively regulate phagocytosis by mammalian cells, often by aVecting ERK and/or PKC signalling, these include phosphatidylinositol 3-kinase (PI3-K) and protein kinase A (PKA) (see for example, Allen et al., 2005; AronoV et al., 2005; Lutz and Correll, 2003). PI-3K and PKA are ubiquitous enzymes, PI3-K phosphorylates phosphoinositides at the 3⬘ position of the inositol ring and PKA is controlled by the levels of cyclic AMP (cAMP) in cells; both of these enzymes have a number of downstream signalling targets. In our quest to better understand the molecular processes that regulate the innate immune response of snail defence cells, we explored the possibility that PI3-K and/or PKA pathways comprise part of the regulatory machinery that controls phagocytosis in L. stagnalis haemocytes. To our knowledge, this research note is the Wrst report to demonstrate a potential role for PI3-K in phagocytosis by molluscan haemocytes.
L.D. Plows et al. / Journal of Invertebrate Pathology 91 (2006) 74–77
lowest dose tested (0.1 M), LY294002 signiWcantly reduced phagocytic activity by 22% compared to controls (P 6 0.001); however at the highest concentration used (10 M) the eVect of this inhibitor was much more marked (62% reduction compared to controls). These Wndings are consistent with PI3-K modulating phagocytosis by L. stagnalis haemocytes. LY294002 is often used to block PI3-K activation at higher concentrations than those employed in the present study (e.g., 20–60 M in Drosophila Schneider cells, Kim et al., 2004); however, 0.1–10 M LY294002 was chosen here to limit the possibility of the inhibitor aVecting other signalling pathways. PI3-K has also been implicated in phagocytosis by ascidian haemocytes (Ishikawa et al., 2000), and the modulation of medXy haemocyte cell shape changes and LPS endocytosis (Soldatos et al., 2003). Moreover, in haemocytes derived from the bivalve mollusc Mytilus galloprovincialis, inhibition of PI3-K reduced bacterial killing, indicating a role for this lipid kinase in the innate immune response (Canesi et al., 2002). Our results support the possibility that the reduction in bacterial killing observed by these authors may at least in part have been due to a reduction in haemocyte phagocytic activity. The phospho-
Phagocytic activity (%)
A 120 100 ***
80
***
60
***
40 20 0
Control
0.1µM
1µM
10µM
LY 294002
B 120 Phagocytic activity (%)
Experiments were performed with primary haemocytes freshly extracted from laboratory cultures of adult L. stagnalis. These cultures were reared from eggs produced by snails purchased from Blades Biologicals (Edenbridge, UK). Juvenile snails were reared 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 °C. Water for maintaining the snails was Wltered through a Brimak/carbon Wltration unit (Silverline, Winkleigh, UK) and was changed weekly. Snails were fed fresh lettuce ad libitum. Adult L. stagnalis were rinsed with distilled water and haemolymph was extracted from individual snails by head–foot retraction (Sminia, 1972). Haemolymph from several 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, and 4 mM CaCl2, pH 7.8, sterilised through a 0.22 m disposable Wlter; 1 part SSS:2 parts haemolymph; Adema et al., 1994). Monolayers were then prepared by allowing haemocytes to adhere to individual wells (200 l diluted haemolymph, containing t6 £ 105 haemocytes, well¡1) of a 96-well cell culture plate (Nunc) for 30 min at room temperature before being washed three times with SSS. Haemocytes were subsequently incubated with either the PKA inhibitor KT5720 (50–100 nM; Calbiochem, Nottingham, UK), the PI3-K inhibitor LY294002 (0.1–10 M; Calbiochem), or vehicle (DMSO), for 30 min. The inhibitor concentrations chosen were similar to those used in other studies (see for example Soldatos et al., 2003) and they span the Ki value for each inhibitor. Next, FITC-conjugated Escherichia coli bioparticles (6 £ 106 per well; Sigma–Aldrich, Poole, UK) were delivered to haemocytes in the presence of inhibitors (or vehicle) for 1 h at room temperature in a dark chamber. Bioparticles were then removed and 2% (w/ v) trypan blue (Sigma–Aldrich) added to each of the wells for 2 min to quench extracellular Xuorescence. Intracellular Xuorescence (derived from phagocytosed bioparticles) was subsequently quantiWed using a Fluorstar Optima microplate spectroXuorimeter (BMG Labtech, Aylesbury, UK). Trypan blue dye exclusion assays were used to visually assess potentially lethal eVects of the inhibitors on cells and neither inhibitor appeared to aVect the viability of the haemocytes at any of the concentrations used. Raw data from three independent experiments were analysed by one-way analysis of variance (ANOVA) and post hoc multiple comparison tests (Tukey) using the statistical software package SPSS. LY294002, a potent and highly selective inhibitor of PI3-K, has been used extensively in studies on mammalian cells. In addition, this inhibitor has been used to deWne the role of PI3-K proteins in Drosophila Schneider cells (Kim et al., 2004), the blowXy Phormia regina (Maniere et al., 2004) and the mosquito Aedes aegypti (Hansen et al., 2005; Riehle and Brown, 1999). When haemocytes were exposed to LY294002, a dose-responsive inhibition of phagocytosis was observed (P 6 0.001; Fig. 1A). At the
75
100 80 60 40 20 0 Control
50nM
100nM KT 5720
Fig. 1. EVects of PI3-K or PKA inhibitors on phagocytosis by L. stagnalis haemocytes. (A) LY294002, (B) KT5720, or vehicle (shown as controls), were pre-incubated with haemocytes prior to challenge with Xuorescent E. coli bioparticles. Uptake of bioparticles was then determined by spectroXuorimetry. Values shown are means (§SEM; n D 6). ***P 6 0.001 when compared to control values.
76
L.D. Plows et al. / Journal of Invertebrate Pathology 91 (2006) 74–77
inositide signalling system, of which PI3-K is an integral component, is known to be a potent modulator of immune responses in mammalian defence cells (reviewed by Stephens et al., 2002). Given that PI3-K can activate PKC in response to bacterial lipopolysaccharide (Monick et al., 2000) and that PKC modulates phagocytosis in molluscan haemocytes (Humphries and Yoshino, 2003; Plows et al., 2004), the possibility that PI3-K activates PKC to drive the phagocytic response in molluscs warrants investigation. KT5720, a speciWc inhibitor of cAMP-dependent PKA, has also been employed in studies on invertebrates; for example, it has been used to explore ion-uptake in the crab Chasmagnathus granulatus (Halperin et al., 2004) and glutamate up-take in the marine mollusc Aplysia (Khabour et al., 2004). When employed in the present study at concentrations of 50 and 100 nM, KT5720 only marginally reduced mean levels of phagocytosis by 9 and 13%, respectively. These observed changes were not signiWcantly diVerent from controls, suggesting that PKA does not play a role in regulating phagocytosis by L. stagnalis haemocytes. This contrasts results from a study with haemocytes from the oyster Crassostrea gigas, which imply a role for PKA in this innate defence response (Lacoste et al., 2001). Other studies indicate that a distinct role for PKA signalling in phagocytosis may not have been conserved through evolution. For example, in mammalian neutrophils, PKA inhibition downregulates phagocytosis (Ydrenius et al., 2000), whereas it is increased in wax moth, Galleria mellonella, haemocytes (Brooks and Dunphy, 2005). Based on current knowledge, that PKA may modulate phagocytosis in haemocytes from some invertebrate species, but not others, seems likely and this may by further inXuenced by the nature of the phagocytic target. The data presented in this research note imply a role for PI3-K, but not PKA, in phagocytosis by haemocytes of L. stagnalis. We now aim to study further the PI3-K pathway in this model snail species. L. stagnalis is host to the avian schistosome Trichobilharzia ocellata, thus knowledge gained concerning the defence response of this snail will allow us to extend our studies to better understand cell signalling in snail–schistosome interactions. In this context, although phagocytosis of bacteria and encapsulation of parasites are diVerent biological responses, the initial recognition events and their eVects on the downstream signalling pathways coordinating cell movements are likely to be similar. Only by furthering our knowledge of the molecular mechanisms that drive invertebrate defence responses in general will we gain a comprehensive understanding of how invading pathogens either survive by subverting such responses, or get eliminated, by the internal defence system. Acknowledgments We are grateful for the Wnancial support of the Royal Society and Kingston University.
References Adema, C.M., Van Deutekom-Mulder, E.C., Van der Knaap, W.P.W., Sminia, T., 1994. Schistosomicidal activities of Lymnaea stagnalis haemocytes: the role of oxygen radicals. Parasitology 109, 479–485. Allen, L.A.H., Allgood, J.A., Han, X.M., Wittine, L.M., 2005. Phosphoinositide 3-kinase regulates actin polymerization during delayed phagocytosis of Helicobacter pylori. J. Leukoc. Biol. 78, 220–230. AronoV, D.M., Canetti, C., Serezani, C.H., Luo, M., Peters-Golden, M., 2005. Cutting edge: Macrophage inhibition by cyclic AMP (cAMP): diVerential roles of protein kinase A and exchange protein directly activated by cAMP-1. J. Immunol. 174, 595–599. Brooks, C.L., Dunphy, G.B., 2005. Protein kinase A aVects Galleria mellonella (Insecta: Lepidoptera) larval haemocyte non-self responses. Immunol. Cell Biol. 83, 150–159. Canesi, L., Betti, M., Ciacci, C., Scarpato, A., Citterio, B., Pruzzo, C., Gallo, G., 2002. Signaling pathways involved in the physiological response of mussel hemocytes to bacterial challenge: the role of stress-activated p38 MAP kinases. Dev. Comp. Immunol. 26, 325–334. Halperin, J., Genovese, G., Tresguerres, M., Luquet, C.M., 2004. Modulation of ion uptake across posterior gills of the crab Chasmagnathus granulatus by dopamine and cAMP. Comp. Biochem. Physiol. A 139, 103–109. Hansen, I.A., Attardo, G.M., Roy, S.G., Raikhel, A.S., 2005. Target of rapamycin-dependent activation of S6 kinase is a central step in the transduction of nutritional signals during egg development in a mosquito. J. Biol. Chem. 280, 20565–20572. Humphries, J.E., Yoshino, T.P., 2001. Protein kinase C regulation of cell spreading in the molluscan Biomphalaria glabrata embryonic (Bge) cell line. Biochim. Biophys. Acta 1540, 243–252. Humphries, J.E., Yoshino, T.P., 2003. Cellular receptors and signal transduction in molluscan haemocytes: connections with the innate immune system of vertebrates. Integr. Comp. Biol. 43, 305–312. Ishikawa, G., Azumi, K., Yokosawa, H., 2000. Involvement of tyrosine kinase and phosphatidylinositol 3-kinase in phagocytosis by ascidian haemocytes. Comp. Biochem. Physiol. A 125, 351–357. Khabour, O., Levenson, J., Lyons, L.C., Kategaya, L.S., Chin, J., Byrne, J.H., Eskin, A., 2004. Coregulation of glutamate uptake and long-term sensitization in Aplysia. J. Neurosci. 24, 8829–8837. Kim, S.E., Cho, J.Y., Kim, K.S., Lee, S.J., Lee, K.H., Choi, K.Y., 2004. Drosophila PI3 kinase and Akt involved in insulin-stimulated proliferation and ERK pathway activation in Schneider cells. Cell. Signal. 16, 1309– 1317. Lacoste, A., Malham, S.K., CueV, A., Poulet, S.A., 2001. Noradrenaline modulates oyster hemocyte phagocytosis via a -adrenergic receptorcAMP signalling pathway. Gen. Comp. Endocrinol. 122, 252–259. Lutz, M.A., Correll, P.H., 2003. Activation of CR3-mediated phagocytosis by MSP requires the RON receptor, tyrosine kinase activity, phosphatidylinositol 3-kinase, and protein kinase C. J. Leukoc. Biol. 73, 802–814. Maniere, G., Rondot, I., Bullesbach, E.E., Gautron, F., Vanhems, E., Delbecque, J.P., 2004. Control of ovarian steroidogenesis by insulin-like peptides in the blowXy (Phormia regina). J. Endocrinol. 181, 147–156. Monick, M.M., Carter, A.B., Flaherty, D.M., Peterson, M.W., Hunninghake, G.W., 2000. Protein kinase C plays a central role in activation of the p42/44 mitogen-activated protein kinase by endotoxin in alveolar macrophages. J. Immunol. 165, 4632–4639. Riehle, M.A., Brown, M.R., 1999. Insulin stimulates ecdysteroid production through a conserved signalling cascade in the mosquito Aedes aegypti. Insect Biochem. Mol. Biol. 29, 855–860. Plows, L.D., Cook, R.T., Davies, A.J., Walker, A.J., 2004. Activation of extracellular signal-regulated kinase is required for phagocytosis by Lymnaea stagnalis haemocytes. Biochim. Biophys. Acta 1692, 25–33. Soldatos, A.N., Metheniti, A., Mamali, I., Lambropoulou, M., Marmaras, V.J., 2003. Distinct LPS-induced signals regulate LPS uptake and morphological changes in medXy haemocytes. Insect Biochem. Mol. Biol. 33, 1075–1084. Sminia, T., 1972. Structure and function of blood and connective tissue cells of the freshwater pulmonate Lymnaea stagnalis studied by elec-
L.D. Plows et al. / Journal of Invertebrate Pathology 91 (2006) 74–77 tron microscopy and enzyme histochemistry. Z. Zellforsch. 130, 497– 526. Stephens, L., Ellson, C., Hawkins, P., 2002. Roles of PI-3Ks in leukocyte chemotaxis and phagocytosis. Curr. Opin. Cell Biol. 14, 203–213. Van der Knaap, W.P.W., Adema, C.M., Sminia, T., 1993. Invertebrate blood cells: morphological and functional aspects of the haemocytes in the pond snail Lymnaea stagnalis. Comp. Haematol. Int. 3, 20–26.
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Walker, A.J., Plows, L.D., 2003. Bacterial lipopolysaccharide modulates protein kinase C signalling in Lymnaea stagnalis haemocytes. Biol. Cell 95, 527–533. Ydrenius, L., Majeed, M., Rasmusson, B.J., Stendahl, O., Sarndahl, E., 2000. Activation of cAMP-dependent protein kinase is necessary for actin rearrangements in human neutrophils during phagocytosis. J. Leukoc. Biol. 67, 520–528.
Research note
Phagocytosis by Lymnaea stagnalis haemocytes: A potential role for phosphatidylinositol 3-kinase but not protein kinase A Louise D. Plows 1, 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 2005; accepted 26 October 2005
Abstract The molecular events that regulate phagocytosis, an important innate immune response, in invertebrate defence cells (haemocytes) are poorly understood. Lymnaea stagnalis haemocytes were used as a model to elucidate the role of cell signalling pathways in phagocytosis by molluscan defence cells. The phosphatidylinositol 3-kinase (PI3-K) inhibitor, LY294002, signiWcantly impaired haemocyte phagocytic activity in a dose-responsive manner with 10 M LY294002 reducing internalization of Xuorescent-conjugated Escherichia coli by 62% (P 6 0.001). In contrast, the protein kinase A (PKA) inhibitor KT5720 was without eVect. Therefore, PI3-K, but not PKA, appears to control phagocytosis by haemocytes in these gastropod molluscs. 2005 Elsevier Inc. All rights reserved. Keywords: Lymnaea stagnalis; Snail; Invertebrate defence; E. coli; Protein kinase A; Phosphatidylinositol 3-kinase
Mobile phagocytic cells called haemocytes play a major part in limiting infection in invertebrates. To date our knowledge of the molecular mechanisms that regulate the defence behaviour of these macrophage-like cells remains fragmentary. This is particularly true for the haemocytes of molluscs which facilitate the removal or sequestration of a range of infectious organisms including bacteria and incompatible parasites (Van der Knaap et al., 1993). Recently, we have identiWed two key signalling enzymes, protein kinase C (PKC) and extracellular signal-regulated kinase (ERK), in freshly collected (primary) haemocytes of the model snail Lymnaea stagnalis (Plows et al., 2004; Walker and Plows, 2003). Our work on these signalling proteins revealed that their activities are modulated by bacterial products and that both pathways are key regulators of the phagocytic response. Other workers have shown that these pathways mediate the spreading behaviour of a haemocyte-like, embryonic cell line (Bge) from the snail Biom*
Corresponding author. Fax: +44 0 20 8547 7562. E-mail address: [email protected] (A.J. Walker). 1 Present address: The Kennedy Institute of Rheumatology, Faculty of Medicine, Imperial College, ARC Building, 1 Aspenlea Road, London W6 8LH, UK. 0022-2011/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2005.10.011
phalaria glabrata, intermediate host to the human schistosome, Schistosoma mansoni (Humphries and Yoshino, 2001). By inhibition assay, primary haemocytes from B. glabrata have also been shown to rely on PKC activity for phagocytosis (Humphries and Yoshino, 2003). A number of other signalling enzymes either positively or negatively regulate phagocytosis by mammalian cells, often by aVecting ERK and/or PKC signalling, these include phosphatidylinositol 3-kinase (PI3-K) and protein kinase A (PKA) (see for example, Allen et al., 2005; AronoV et al., 2005; Lutz and Correll, 2003). PI-3K and PKA are ubiquitous enzymes, PI3-K phosphorylates phosphoinositides at the 3⬘ position of the inositol ring and PKA is controlled by the levels of cyclic AMP (cAMP) in cells; both of these enzymes have a number of downstream signalling targets. In our quest to better understand the molecular processes that regulate the innate immune response of snail defence cells, we explored the possibility that PI3-K and/or PKA pathways comprise part of the regulatory machinery that controls phagocytosis in L. stagnalis haemocytes. To our knowledge, this research note is the Wrst report to demonstrate a potential role for PI3-K in phagocytosis by molluscan haemocytes.
L.D. Plows et al. / Journal of Invertebrate Pathology 91 (2006) 74–77
lowest dose tested (0.1 M), LY294002 signiWcantly reduced phagocytic activity by 22% compared to controls (P 6 0.001); however at the highest concentration used (10 M) the eVect of this inhibitor was much more marked (62% reduction compared to controls). These Wndings are consistent with PI3-K modulating phagocytosis by L. stagnalis haemocytes. LY294002 is often used to block PI3-K activation at higher concentrations than those employed in the present study (e.g., 20–60 M in Drosophila Schneider cells, Kim et al., 2004); however, 0.1–10 M LY294002 was chosen here to limit the possibility of the inhibitor aVecting other signalling pathways. PI3-K has also been implicated in phagocytosis by ascidian haemocytes (Ishikawa et al., 2000), and the modulation of medXy haemocyte cell shape changes and LPS endocytosis (Soldatos et al., 2003). Moreover, in haemocytes derived from the bivalve mollusc Mytilus galloprovincialis, inhibition of PI3-K reduced bacterial killing, indicating a role for this lipid kinase in the innate immune response (Canesi et al., 2002). Our results support the possibility that the reduction in bacterial killing observed by these authors may at least in part have been due to a reduction in haemocyte phagocytic activity. The phospho-
Phagocytic activity (%)
A 120 100 ***
80
***
60
***
40 20 0
Control
0.1µM
1µM
10µM
LY 294002
B 120 Phagocytic activity (%)
Experiments were performed with primary haemocytes freshly extracted from laboratory cultures of adult L. stagnalis. These cultures were reared from eggs produced by snails purchased from Blades Biologicals (Edenbridge, UK). Juvenile snails were reared 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 °C. Water for maintaining the snails was Wltered through a Brimak/carbon Wltration unit (Silverline, Winkleigh, UK) and was changed weekly. Snails were fed fresh lettuce ad libitum. Adult L. stagnalis were rinsed with distilled water and haemolymph was extracted from individual snails by head–foot retraction (Sminia, 1972). Haemolymph from several 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, and 4 mM CaCl2, pH 7.8, sterilised through a 0.22 m disposable Wlter; 1 part SSS:2 parts haemolymph; Adema et al., 1994). Monolayers were then prepared by allowing haemocytes to adhere to individual wells (200 l diluted haemolymph, containing t6 £ 105 haemocytes, well¡1) of a 96-well cell culture plate (Nunc) for 30 min at room temperature before being washed three times with SSS. Haemocytes were subsequently incubated with either the PKA inhibitor KT5720 (50–100 nM; Calbiochem, Nottingham, UK), the PI3-K inhibitor LY294002 (0.1–10 M; Calbiochem), or vehicle (DMSO), for 30 min. The inhibitor concentrations chosen were similar to those used in other studies (see for example Soldatos et al., 2003) and they span the Ki value for each inhibitor. Next, FITC-conjugated Escherichia coli bioparticles (6 £ 106 per well; Sigma–Aldrich, Poole, UK) were delivered to haemocytes in the presence of inhibitors (or vehicle) for 1 h at room temperature in a dark chamber. Bioparticles were then removed and 2% (w/ v) trypan blue (Sigma–Aldrich) added to each of the wells for 2 min to quench extracellular Xuorescence. Intracellular Xuorescence (derived from phagocytosed bioparticles) was subsequently quantiWed using a Fluorstar Optima microplate spectroXuorimeter (BMG Labtech, Aylesbury, UK). Trypan blue dye exclusion assays were used to visually assess potentially lethal eVects of the inhibitors on cells and neither inhibitor appeared to aVect the viability of the haemocytes at any of the concentrations used. Raw data from three independent experiments were analysed by one-way analysis of variance (ANOVA) and post hoc multiple comparison tests (Tukey) using the statistical software package SPSS. LY294002, a potent and highly selective inhibitor of PI3-K, has been used extensively in studies on mammalian cells. In addition, this inhibitor has been used to deWne the role of PI3-K proteins in Drosophila Schneider cells (Kim et al., 2004), the blowXy Phormia regina (Maniere et al., 2004) and the mosquito Aedes aegypti (Hansen et al., 2005; Riehle and Brown, 1999). When haemocytes were exposed to LY294002, a dose-responsive inhibition of phagocytosis was observed (P 6 0.001; Fig. 1A). At the
75
100 80 60 40 20 0 Control
50nM
100nM KT 5720
Fig. 1. EVects of PI3-K or PKA inhibitors on phagocytosis by L. stagnalis haemocytes. (A) LY294002, (B) KT5720, or vehicle (shown as controls), were pre-incubated with haemocytes prior to challenge with Xuorescent E. coli bioparticles. Uptake of bioparticles was then determined by spectroXuorimetry. Values shown are means (§SEM; n D 6). ***P 6 0.001 when compared to control values.
76
L.D. Plows et al. / Journal of Invertebrate Pathology 91 (2006) 74–77
inositide signalling system, of which PI3-K is an integral component, is known to be a potent modulator of immune responses in mammalian defence cells (reviewed by Stephens et al., 2002). Given that PI3-K can activate PKC in response to bacterial lipopolysaccharide (Monick et al., 2000) and that PKC modulates phagocytosis in molluscan haemocytes (Humphries and Yoshino, 2003; Plows et al., 2004), the possibility that PI3-K activates PKC to drive the phagocytic response in molluscs warrants investigation. KT5720, a speciWc inhibitor of cAMP-dependent PKA, has also been employed in studies on invertebrates; for example, it has been used to explore ion-uptake in the crab Chasmagnathus granulatus (Halperin et al., 2004) and glutamate up-take in the marine mollusc Aplysia (Khabour et al., 2004). When employed in the present study at concentrations of 50 and 100 nM, KT5720 only marginally reduced mean levels of phagocytosis by 9 and 13%, respectively. These observed changes were not signiWcantly diVerent from controls, suggesting that PKA does not play a role in regulating phagocytosis by L. stagnalis haemocytes. This contrasts results from a study with haemocytes from the oyster Crassostrea gigas, which imply a role for PKA in this innate defence response (Lacoste et al., 2001). Other studies indicate that a distinct role for PKA signalling in phagocytosis may not have been conserved through evolution. For example, in mammalian neutrophils, PKA inhibition downregulates phagocytosis (Ydrenius et al., 2000), whereas it is increased in wax moth, Galleria mellonella, haemocytes (Brooks and Dunphy, 2005). Based on current knowledge, that PKA may modulate phagocytosis in haemocytes from some invertebrate species, but not others, seems likely and this may by further inXuenced by the nature of the phagocytic target. The data presented in this research note imply a role for PI3-K, but not PKA, in phagocytosis by haemocytes of L. stagnalis. We now aim to study further the PI3-K pathway in this model snail species. L. stagnalis is host to the avian schistosome Trichobilharzia ocellata, thus knowledge gained concerning the defence response of this snail will allow us to extend our studies to better understand cell signalling in snail–schistosome interactions. In this context, although phagocytosis of bacteria and encapsulation of parasites are diVerent biological responses, the initial recognition events and their eVects on the downstream signalling pathways coordinating cell movements are likely to be similar. Only by furthering our knowledge of the molecular mechanisms that drive invertebrate defence responses in general will we gain a comprehensive understanding of how invading pathogens either survive by subverting such responses, or get eliminated, by the internal defence system. Acknowledgments We are grateful for the Wnancial support of the Royal Society and Kingston University.
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