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Involvement of tyrosine kinase and phosphatidylinositol 3-kinase in phagocytosis by ascidian hemocytes
Comparative Biochemistry and Physiology Part A 125 (2000) 351 – 357
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Involvement of tyrosine kinase and phosphatidylinositol 3-kinase in phagocytosis by ascidian hemocytes Go Ishikawa, Kaoru Azumi *, Hideyoshi Yokosawa Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Hokkaido Uni6ersity, Sapporo 060 -0812, Japan Received 7 September 1999; received in revised form 19 January 2000; accepted 31 January 2000
Abstract It has been proposed that protein tyrosine phosphorylation plays important roles in signal transduction in mammalian T- and B-cells and monocytes. During our investigations on the ascidian host defense system, we have shown that the monoclonal antibody A74 strongly inhibits both phagocytosis of sheep red blood cells (SRBCs) by hemocytes and hemocyte aggregation, and that the A74 antigen protein has two immunoreceptor tyrosine-based activation motifs and several other motifs that are thought to function in signal transduction in mammals. In this study, we found that the A74 antibody strongly inhibited phagocytosis by ascidian hemocytes of yeast cells, as strongly as that of SRBCs, but not that of latex beads. We also found that herbimycin A and an erbstatin analog, tyrosine kinase inhibitors, and wortmannin, a specific inhibitor for phosphatidylinositol 3-kinase (PI3-kinase), inhibited the phagocytosis of yeast cells. We investigated which hemocyte proteins were specifically tyrosine-phosphorylated during phagocytosis by ascidian hemocytes and found that a protein with a molecular mass of 100 kDa was specifically tyrosine-phosphorylated upon phagocytosis; its tyrosine phosphorylation was inhibited by the A74 antibody. These results strongly suggest that both tyrosine kinase and PI3-kinase play important roles in phagocytosis by ascidian hemocytes. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Ascidian; Defense system; Halocynthia roretzi; Hemocyte; Phagocytosis; Tyrosine kinase; Tyrosine phosphorylation; Phosphatidylinositol 3-kinase (PI3-kinase)
1. Introduction The innate immune system has been proposed to play an important role in host defense in mammals and interact with the acquired (or adaptive) immune system (Fearon, 1997; Medzhitov and Janeway, 1997; Hoffmann et al., 1999). The former system is phylogenetically conserved and also functions in host defense in invertebrates. Ascidians occupy a phylogenetic position between * Corresponding author. Tel.: + 81-11-7063917; fax: +8111-7064900. E-mail address: [email protected] (K. Azumi)
vertebrates and true invertebrates, and are therefore considered to be ancestors of vertebrates. In the ascidian Halocynthia roretzi, hemocytes undergo several cellular defense reactions including phagocytosis and hemocyte aggregation (Azumi and Yokosawa, 1996). Monoclonal antibodies that inhibit the cellular defense reactions in H. roretzi have been utilized to determine the hemocyte membrane-bound molecules that are involved in these reactions (Azumi and Yokosawa, 1996). The monoclonal antibody A74 strongly inhibits both phagocytosis against sheep red blood cells (SRBCs) by H. roretzi hemocytes and hemocyte aggregation. We purified the A74 antigen protein
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from H. roretzi hemocytes and found that it is a novel membrane glycoprotein with a molecular mass of 160 kDa (Takahashi et al., 1995). Through cDNA cloning of the A74 antigen protein, it was found that the A74 protein has two immunoreceptor tyrosine-based activation motifs (ITAMs) and several other motifs that have been proposed to function in mammalian signal transduction mechanisms (Takahashi et al., 1997). The ITAM has been reported to play an important role in signal transduction in mammalian T-cell antigen receptor (TCR), B-cell antigen receptor (BCR), and Fc receptors (FcRs) through protein tyrosine phosphorylation, which is essential for signal transduction in various cellular events (Weiss and Littman, 1994; Howe and Weiss, 1995). We have already reported that several cellular proteins, including the A74 protein, were tyrosine-phosphorylated during aggregation of H. roretzi hemocytes (Takahashi et al., 1997). In this study, we found that phagocytosis by H. roretzi hemocytes is inhibited by tyrosine kinase inhibitors and by an inhibitor specific for phosphatidylinositol 3-kinase (PI3-kinase). We also found that a certain hemocyte protein was specifically tyrosine-phosphorylated during phagocytosis. Thus, both tyrosine kinase and PI3-kinase may play important roles in phagocytosis by ascidian hemocytes. This is the first report on the involvement of tyrosine phosphorylation and PI3kinase in phagocytosis by invertebrate hemocytes.
2. Materials and methods
2.1. Materials Latex beads (5.7 mm in diameter), wortmannin, phenylmethanesulfonyl fluoride (PMSF), highmolecular mass standards for SDS-polyacrylamide gel electrophoresis (SDS – PAGE) and diisopropylfluorophosphate (DFP) were purchased from Sigma (USA). Herbimycin A and the erbstatin analog were obtained from LC Laboratories (USA). Preimmune mouse immunoglobulin G (IgG), anti-phosphotyrosine monoclonal antibody (PY20) and peroxidase-conjugated antimouse IgG were obtained from Chemicon International, Leinco Technologies (USA) and Dako (Denmark), respectively. ECL, the enhanced chemiluminescence system, and leupeptin were purchased from Amersham (UK) and Pep-
tide Institute (Japan), respectively. Yeast cells (W303D) were provided by Dr A. Toh-e of Tokyo University.
2.2. Preparation of H. roretzi hemocytes and plasma Hemocytes and plasma of the solitary ascidian H. roretzi were prepared as described previously (Takahashi et al., 1994), and the hemocytes were suspended in Ca2 + -, Mg2 + -free Herbst’s artificial sea water (F-HASW; 450 mM NaCl, 9.4 mM KCl, 32 mM Na2SO4 and 3.2 mM NaHCO3, pH 7.6).
2.3. Assay for phagocytosis Yeast cells were stained with 0.4% Congo-red, autoclaved, and suspended in F-HASW (2× 107 cells/ml). Latex beads were washed and suspended in F-HASW (2× 107 particles/ml). After an H. roretzi hemocyte suspension (1×107 cells/ml) in a volume of 20 ml in the presence or absence of 20 ml of antibody or kinase inhibitor solution was allowed to stand for 30 min on ice, 20 ml of a suspension of yeast cells or latex beads was added, and the mixture was incubated for 1 h at 20°C. The phagocytosis by hemocytes was stopped by adding 20 ml of 100 mM diethyldithiocarbamate, a metal-chelating agent. After the hemocytes were transferred to a slide glass and stained with Nile blue dye, the phagocytic activity was measured under a Nikon phase contrast microscope; 200 hemocytes were inspected to check whether the hemocytes had ingested materials. A hemocyte that had ingested at least one material was counted as a positive one. The degree of phagocytosis was expressed as the ratio of the number of positive hemocytes to the number of total hemocytes.
2.4. Western blot analysis After H. roretzi hemocytes (4×106 cells) were stimulated by addition of yeast cells (4× 107 cells) at 20°C for an appropriate time, they were collected by centrifugation at 19 000× g for 15 s and immediately frozen in liquid N2. The frozen cell pellet was sonicated in 100 ml of SDS sample buffer containing 5 mM EDTA, 5 mM leupeptin, 2 mM DFP, 2 mM PMSF, 2 mM Na3VO4, 50 mM NaF and 5% 2-mercaptoethanol. The cell
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lysate was then boiled at 90°C for 5 min. Proteins were separated by SDS – PAGE on a 7.5% gel (Laemmli, 1970) and subjected to Western blotting (Towbin et al., 1979) using anti-phosphotyrosine antibody PY20 as a primary antibody and peroxidase-conjugated anti-mouse IgG as a secondary antibody. Tyrosine phosphorylation of hemocyte proteins was measured by detection using ECL, and the bands were visualized with X-ray film.
3. Results
3.1. Effect of A74 antibody on phagocytosis by H. roretzi hemocytes It has previously been reported that the A74 antibody strongly inhibits phagocytosis of SRBCs by H. roretzi hemocytes (Takahashi et al., 1995). As shown in Fig. 1A, the A74 antibody also inhibited the phagocytosis of another target material, yeast cells, in a concentration-dependent manner. The A74 antibody showed almost 50 and 80% inhibition at concentrations of 1 mg/ml and 10 mg/ml, respectively, while control mouse IgG did not inhibit the phagocytosis. On the other hand, the A74 antibody scarcely inhibited the phagocytosis of a non-cellular target material, latex beads (Fig. 1B). It should be noted that,
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under the conditions used, 30% of total hemocytes ingested at least one yeast cell or latex bead in the absence of A74 antibody or control IgG.
3.2. Effects of kinase inhibitors on phagocytosis by H. roretzi hemocytes Next, we investigated whether protein kinase and PI3-kinase are involved in phagocytosis of yeast cells by H. roretzi hemocytes. The effects of kinase inhibitors on the phagocytosis are shown in Fig. 2. In these experiments, dimethylsulfoxide (DMSO) was used as a solvent of the inhibitors (final DMSO concentrations were 2.85 and 0.006% in Fig. 2A and B, respectively), and 29% of total hemocytes ingested at least one yeast cell in the absence or presence of 0.006% DMSO, while 27% did so in the presence of 2.85% DMSO (2.85% DMSO showed 7% inhibition against phagocytosis). As shown in Fig. 2A, the erbstatin analog and herbimycin A, tyrosine kinase inhibitors, both inhibited the phagocytosis of yeast cells. The erbstatin analog inhibited phagocytosis in a concentration-dependent manner and showed almost 90% inhibition at a concentration of 50 mM. Herbimycin A also inhibited phagocytosis in a concentration-dependent manner, but only 50% inhibition was observed even at a concentration of 50 mM. These results suggest that at least two tyrosine kinases, an erbstatin analog-sensitive one
Fig. 1. Effect of the A74 antibody on phagocytosis by H. roretzi hemocytes of yeast cells or latex beads. Phagocytosis of yeast cells (A) or latex beads (B) in the presence of the A74 antibody or control mouse IgG was measured. Approximately 200 hemocytes were inspected to check whether the hemocytes had ingested materials, and a hemocyte that had ingested at least one material was counted as a positive one. The degree of phagocytosis (%) was expressed as the ratio of the number of positive hemocytes to that of total ones. The concentrations of antibody and control IgG in B were 5 mg/ml. Bars are mean 9 S.D.; n =3.
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Fig. 2. Effects of kinase inhibitors on phagocytosis of yeast cells by H. roretzi hemocytes. The hemocytes were previously incubated for 30 min at 4°C in the presence or absence of herbimycin A (open bar in A), erbstatin analog (closed bar in A) and wortmannin (B), and yeast cells were then added to the hemocyte suspension, and the mixture was incubated at 20°C for 1 h. It should be noted that 27% of total hemocytes ingested at least one yeast cell in the absence of inhibitors but the presence of 2.85% dimethylsulfoxide (DMSO) (A), while 29% did in the presence of only 0.006% DMSO (B), as well as in the absence of DMSO. Bars in A mean9 S.D. (n =3) and the results shown in B are the means of the results of duplicate experiments.
and a herbimycin-sensitive one, are involved in phagocytosis by H. roretzi hemocytes. In the inhibition experiment with wortmannin, a specific inhibitor for PI3-kinase, the phagocytosis of yeast cells was inhibited, and 50% inhibition was observed at a concentration as low as 0.06 mM (Fig. 2B), suggesting that a PI3-kinase-like enzyme is involved in phagocytosis by H. roretzi hemocytes.
3.3. Tyrosine phosphorylation of hemocyte proteins during phagocytosis by H. roretzi hemocytes To address the question as to whether tyrosine phosphorylation of specific hemocyte proteins is involved in signal transduction during phagocytosis by H. roretzi hemocytes, we analyzed the tyrosine phosphorylation upon addition of yeast cells to the hemocytes (Fig. 3A). In a control experiment without yeast cells, H. roretzi hemocytes gradually underwent aggregation, and tyrosine phosphorylation of several proteins of 160, 110, 86, 75 and 56 kDa was observed 5 – 10 min after initiation of incubation. On the other hand, upon addition of yeast cells to the hemocytes, the above proteins of 160, 110, 86 and 75 kDa were quickly tyrosine-phosphorylated, and tyrosine phosphorylation of a new protein of 100 kDa was
Fig. 3. Tyrosine phosphorylation during phagocytosis by H. roretzi hemocytes. The hemocytes were collected in a process of phagocytosis of yeast cells and subjected to SDS – PAGE, followed by Western blotting with the anti-phosphotyrosine antibody (A). Proteins were stained with Coomassie brilliant blue (B).
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4. Discussion
Fig. 4. Effects of the A74 antibody and kinase inhibitors on tyrosine phosphorylation during phagocytosis by H. roretzi hemocytes. After the hemocytes were pretreated with control mouse IgG (10 mg/ml, lane 2), the A74 antibody (10 mg/ml, lane 3), wortmannin (0.1 mM, lane 4), herbimycin A (1 mM, lane 5) and erbstatin analog (1 mM, lane 6), or without reagent (lane 1) at 4°C for 30 min, yeast cells were added to each hemocyte suspension and the mixture was incubated at 20°C for 10 min. The hemocytes in each case were collected and subjected to SDS – PAGE, followed by Western blotting with the anti-phosphotyrosine antibody.
also observed. In addition, it should be noted that the tyrosine phosphorylation of the A74 protein (160 kDa) was detected in both control and phagocytosis experiments. Next, we investigated whether the A74 protein, herbimycin-sensitive and erbstatin analog-sensitive tyrosine kinases, and PI3-kinase function upstream of the above tyrosine phosphorylation observed during phagocytosis. The effects of the A74 antibody, two tyrosine kinase inhibitors (herbimycin A and the erbstatin analog) and the PI3-kinase inhibitor (wortmannin) on the yeastinduced tyrosine phosphorylation of hemocyte proteins are shown in Fig. 4. Pretreatment with the A74 antibody enhanced the tyrosine phosphorylation of the A74 protein (160 kDa) and slightly enhanced the tyrosine phosphorylation of 110-, 86-, 75- and 56-kDa proteins. However, the tyrosine phosphorylation of the 100-kDa protein, which appears to be a specific event for the phagocytosis of yeast cells, was strongly inhibited by pretreatment with the A74 antibody. The three kinase inhibitors used had little effect on the tyrosine phosphorylation of hemocyte proteins, including the 100-kDa protein, at concentrations where at least 50% inhibition of phagocytosis was observed (Fig. 2). These results suggest that the A74 protein may function upstream of the tyrosine phosphorylation of the 100-kDa protein and that the two putative protein kinases and PI3-kinase may work downstream of the above tyrosine phosphorylation.
H. roretzi hemocytes undergo phagocytosis of SRBCs, yeast cells, latex beads and Escherichia coli in vitro (Ohtake et al., 1994; Takahashi et al., 1995; Azumi and Yokosawa, 1996). To determine the molecules that are involved in phagocytosis by H. roretzi hemocytes, we prepared the monoclonal antibody A74, which strongly inhibited phagocytosis of SRBCs and recognized the 160kDa antigen protein (Takahashi et al., 1995). In this study, we first showed that the A74 antibody also inhibited the phagocytosis of another cellular target material, yeast cells, together with SRBCs, but not that of a non-cellular target material, latex beads (Fig. 1). These results suggest that the A74 protein can interact with common surface materials on SRBCs and yeast cells. The A74 protein has two ITAMs, three src homology 2 (SH2) binding motifs, one src homology 3 (SH3) binding motif and two pairs of trithreonine (TTT) motifs, all of which have been proposed to function in signal transduction in mammals (Takahashi et al., 1997). We have already demonstrated that the ITAMs expressed as glutathione S-transferase-fusion proteins were tyrosine-phosphorylated by human c-Src kinase in vitro (Takahashi et al., 1997). These results led us to speculate that the ITAMs in the A74 protein are involved in the initial stage of signal transduction through tyrosine phosphorylation. To obtain in vivo evidence for the involvement of tyrosine phosphorylation in signal transduction during phagocytosis by H. roretzi hemocytes, we next investigated the effects of kinase inhibitors on phagocytosis. In mammals, the involvement of protein tyrosine kinases in FcgR-mediated signaling events has been revealed by using specific tyrosine kinase inhibitors. Several inhibitors, including herbimycin A and erbstatin, blocked phagocytosis by monocytes and macrophages and also inhibited tyrosine phosphorylation of various proteins involved in phagocytosis (Greenberg et al., 1993; Strzelecka et al., 1997). In the case of phagocytosis by ascidian hemocytes, two tyrosine kinase inhibitors (herbimycin A and the erbstatin analog) inhibited phagocytosis, but the sensitivities against the two inhibitors seemed different (Fig. 2A). Thus, it is possible that at least two different tyrosine kinases, an erbstatin analog-sensitive one and a herbimycin-sensitive one, may be involved in phagocytosis by H. roretzi hemocytes.
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In human monocytes, the involvement of PI3-kinase for FcgR-mediated phagocytosis was revealed by studies using wortmannin, a specific PI3-kinase inhibitor; the inhibitor blocked phagocytosis (Ninomiya et al., 1994). We also found that wortmannin inhibited phagocytosis of yeast cells by H. roretzi hemocytes (Fig. 2B), suggesting that a PI3-kinase-like enzyme is involved in phagocytosis by ascidian hemocytes. Next, we analyzed tyrosine phosphorylation during phagocytosis by Western blotting using an antiphosphotyrosine antibody to determine which hemocyte proteins were tyrosine phosphorylated. Immediately after the addition of target materials, the tyrosine phosphorylation of proteins of 160, 110, 86 and 75 kDa was enhanced, and a new protein of 100 kDa was tyrosine-phosphorylated (Fig. 3). Tyrosine phosphorylation of the above proteins was still observed in the presence of the three kinase inhibitors at concentrations where the inhibitors can suppress phagocytosis by 50% (Fig. 4). These results suggest that the above tyrosine phosphorylation may occur at an early stage of phagocytosis and that the two putative tyrosine kinases and the PI3-kinase-like protein may work downstream of the above event. Interestingly, we found that the A74 antibody, which can inhibit both interactions of hemocytes with conspecific hemocytes (aggregation) and with foreign materials (phagocytosis), enhanced tyrosine phosphorylation of all of the above proteins (including the A74 protein) except for the 100-kDa protein (Fig. 4). Considering the above results together with the fact that tyrosine residues in the cytoplasmic parts of FcgRs became phosphorylated upon activation of the receptors in human monocytes by their cross-linking with the specific antibodies (Duchemin et al., 1994), it can be inferred that the A74 protein functions as a signal-recognition and -transduction molecule in phagocytosis by ascidian hemocytes. With respect to the role of the 100-kDa protein in phagocytosis, the fact that the A74 antibody strongly inhibited phagocytosis-specific tyrosine phosphorylation of the 100-kDa protein (Fig. 4) led us to speculate that the 100-kDa protein, together with the A74 protein, functions at an early stage of phagocytosis, such as hemocyte recognition of foreign materials, and transduces their signals downstream through its tyrosine phosphorylation.
Further studies on identification of the 100-kDa protein and the tyrosine kinase functioning in its tyrosine phosphorylation, as well as elucidation of the roles of the two putative tyrosine kinases and the PI3-kinase functioning at a later stage of phagocytosis, are needed to obtained detailed knowledge on the mechanisms of phagocytosis by ascidian hemocytes. Acknowledgements This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan. References Azumi, K., Yokosawa, H., 1996. Humoral factors and cellular reactions in the biological defense of the ascidian H. roretzi. In: So¨derha¨ll, K., Iwanaga, S., Vasta, G. (Eds.), New Directions in Invertebrate Immunology. SOS Publications, Fair Haven, pp. 43 – 53. Duchemin, A.-M., Ernst, L.K., Anderson, C.L., 1994. Clustering of the high affinity Fc receptor for immunoglobulin G (FcgRI) results in phosphorylation of its associated g-chain. J. Biol. Chem. 269, 12111 – 12117. Fearon, D.T., 1997. Seeking wisdom in innate immunity. Nature 388, 323 – 324. Greenberg, S., Chang, P., Silverstein, S.C., 1993. Tyrosine phosphorylation is required for Fc receptormediated phagocytosis in mouse macrophages. J. Exp. Med. 177, 529 – 534. Hoffmann, J.A., Kafatos, F.C., Janeway, C.A. Jr, Ezekowitz, R.A.B., 1999. Phylogenetic perspectives in innate immunity. Science 284, 1313 – 1318. Howe, L.R., Weiss, A., 1995. Multiple kinases mediate T-cell-receptor signaling. Trends Biochem. Sci. 20, 59 – 64. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685. Medzhitov, R., Janeway, C.A. Jr, 1997. Innate immunity: the virtues of a non-clonal system of recognition. Cell 91, 295 – 298. Ninomiya, N., Hazeki, K., Fukui, Y., Seya, T., Okada, T., Hazeki, O., Ui, M., 1994. Involvement of phosphatidylinositol 3-kinase in Fcg receptor signaling. J. Biol. Chem. 269, 22732 – 22737. Ohtake, S., Abe, T., Shishikura, F., Tanaka, K., 1994. The phagocytes in hemolymph of Halocynthia roretzi and their phagocytic activity. Zool. Sci. 11, 681 – 691.
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Strzelecka, A., Kwiatkowska, K., Sobota, A., 1997. Tyrosine phosphorylation and Fcg receptor-mediated phagocytosis. FEBS Lett. 400, 11–14. Takahashi, H., Azumi, K., Yokosawa, H., 1994. Hemocyte aggregation in the solitary ascidian, Halocynthia roretzi: plasma factors, magnesium ion, and Met-Lys-bradykinin induce the aggregation. Biol. Bull. 186, 247–253. Takahashi, H., Azumi, K., Yokosawa, H., 1995. A novel membrane glycoprotein involved in ascidian hemocyte aggregation and phagocytosis. Eur. J. Biochem. 233, 778–783.
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Takahashi, H., Ishikawa, G., Ueki, K., Azumi, K., Yokosawa, H., 1997. Cloning and tyrosine phosphorylation of a novel invertebrate immunocyte protein containing immunoreceptor tyrosine-based activation motifs. J. Biol. Chem. 272, 32006 – 32010. Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350 – 4354. Weiss, A., Littman, D.R., 1994. Signal transduction by lymphocyte antigen receptors. Cell 76, 263 – 274.
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Involvement of tyrosine kinase and phosphatidylinositol 3-kinase in phagocytosis by ascidian hemocytes Go Ishikawa, Kaoru Azumi *, Hideyoshi Yokosawa Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Hokkaido Uni6ersity, Sapporo 060 -0812, Japan Received 7 September 1999; received in revised form 19 January 2000; accepted 31 January 2000
Abstract It has been proposed that protein tyrosine phosphorylation plays important roles in signal transduction in mammalian T- and B-cells and monocytes. During our investigations on the ascidian host defense system, we have shown that the monoclonal antibody A74 strongly inhibits both phagocytosis of sheep red blood cells (SRBCs) by hemocytes and hemocyte aggregation, and that the A74 antigen protein has two immunoreceptor tyrosine-based activation motifs and several other motifs that are thought to function in signal transduction in mammals. In this study, we found that the A74 antibody strongly inhibited phagocytosis by ascidian hemocytes of yeast cells, as strongly as that of SRBCs, but not that of latex beads. We also found that herbimycin A and an erbstatin analog, tyrosine kinase inhibitors, and wortmannin, a specific inhibitor for phosphatidylinositol 3-kinase (PI3-kinase), inhibited the phagocytosis of yeast cells. We investigated which hemocyte proteins were specifically tyrosine-phosphorylated during phagocytosis by ascidian hemocytes and found that a protein with a molecular mass of 100 kDa was specifically tyrosine-phosphorylated upon phagocytosis; its tyrosine phosphorylation was inhibited by the A74 antibody. These results strongly suggest that both tyrosine kinase and PI3-kinase play important roles in phagocytosis by ascidian hemocytes. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Ascidian; Defense system; Halocynthia roretzi; Hemocyte; Phagocytosis; Tyrosine kinase; Tyrosine phosphorylation; Phosphatidylinositol 3-kinase (PI3-kinase)
1. Introduction The innate immune system has been proposed to play an important role in host defense in mammals and interact with the acquired (or adaptive) immune system (Fearon, 1997; Medzhitov and Janeway, 1997; Hoffmann et al., 1999). The former system is phylogenetically conserved and also functions in host defense in invertebrates. Ascidians occupy a phylogenetic position between * Corresponding author. Tel.: + 81-11-7063917; fax: +8111-7064900. E-mail address: [email protected] (K. Azumi)
vertebrates and true invertebrates, and are therefore considered to be ancestors of vertebrates. In the ascidian Halocynthia roretzi, hemocytes undergo several cellular defense reactions including phagocytosis and hemocyte aggregation (Azumi and Yokosawa, 1996). Monoclonal antibodies that inhibit the cellular defense reactions in H. roretzi have been utilized to determine the hemocyte membrane-bound molecules that are involved in these reactions (Azumi and Yokosawa, 1996). The monoclonal antibody A74 strongly inhibits both phagocytosis against sheep red blood cells (SRBCs) by H. roretzi hemocytes and hemocyte aggregation. We purified the A74 antigen protein
1095-6433/00/$ - see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 ( 0 0 ) 0 0 1 6 5 - 3
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from H. roretzi hemocytes and found that it is a novel membrane glycoprotein with a molecular mass of 160 kDa (Takahashi et al., 1995). Through cDNA cloning of the A74 antigen protein, it was found that the A74 protein has two immunoreceptor tyrosine-based activation motifs (ITAMs) and several other motifs that have been proposed to function in mammalian signal transduction mechanisms (Takahashi et al., 1997). The ITAM has been reported to play an important role in signal transduction in mammalian T-cell antigen receptor (TCR), B-cell antigen receptor (BCR), and Fc receptors (FcRs) through protein tyrosine phosphorylation, which is essential for signal transduction in various cellular events (Weiss and Littman, 1994; Howe and Weiss, 1995). We have already reported that several cellular proteins, including the A74 protein, were tyrosine-phosphorylated during aggregation of H. roretzi hemocytes (Takahashi et al., 1997). In this study, we found that phagocytosis by H. roretzi hemocytes is inhibited by tyrosine kinase inhibitors and by an inhibitor specific for phosphatidylinositol 3-kinase (PI3-kinase). We also found that a certain hemocyte protein was specifically tyrosine-phosphorylated during phagocytosis. Thus, both tyrosine kinase and PI3-kinase may play important roles in phagocytosis by ascidian hemocytes. This is the first report on the involvement of tyrosine phosphorylation and PI3kinase in phagocytosis by invertebrate hemocytes.
2. Materials and methods
2.1. Materials Latex beads (5.7 mm in diameter), wortmannin, phenylmethanesulfonyl fluoride (PMSF), highmolecular mass standards for SDS-polyacrylamide gel electrophoresis (SDS – PAGE) and diisopropylfluorophosphate (DFP) were purchased from Sigma (USA). Herbimycin A and the erbstatin analog were obtained from LC Laboratories (USA). Preimmune mouse immunoglobulin G (IgG), anti-phosphotyrosine monoclonal antibody (PY20) and peroxidase-conjugated antimouse IgG were obtained from Chemicon International, Leinco Technologies (USA) and Dako (Denmark), respectively. ECL, the enhanced chemiluminescence system, and leupeptin were purchased from Amersham (UK) and Pep-
tide Institute (Japan), respectively. Yeast cells (W303D) were provided by Dr A. Toh-e of Tokyo University.
2.2. Preparation of H. roretzi hemocytes and plasma Hemocytes and plasma of the solitary ascidian H. roretzi were prepared as described previously (Takahashi et al., 1994), and the hemocytes were suspended in Ca2 + -, Mg2 + -free Herbst’s artificial sea water (F-HASW; 450 mM NaCl, 9.4 mM KCl, 32 mM Na2SO4 and 3.2 mM NaHCO3, pH 7.6).
2.3. Assay for phagocytosis Yeast cells were stained with 0.4% Congo-red, autoclaved, and suspended in F-HASW (2× 107 cells/ml). Latex beads were washed and suspended in F-HASW (2× 107 particles/ml). After an H. roretzi hemocyte suspension (1×107 cells/ml) in a volume of 20 ml in the presence or absence of 20 ml of antibody or kinase inhibitor solution was allowed to stand for 30 min on ice, 20 ml of a suspension of yeast cells or latex beads was added, and the mixture was incubated for 1 h at 20°C. The phagocytosis by hemocytes was stopped by adding 20 ml of 100 mM diethyldithiocarbamate, a metal-chelating agent. After the hemocytes were transferred to a slide glass and stained with Nile blue dye, the phagocytic activity was measured under a Nikon phase contrast microscope; 200 hemocytes were inspected to check whether the hemocytes had ingested materials. A hemocyte that had ingested at least one material was counted as a positive one. The degree of phagocytosis was expressed as the ratio of the number of positive hemocytes to the number of total hemocytes.
2.4. Western blot analysis After H. roretzi hemocytes (4×106 cells) were stimulated by addition of yeast cells (4× 107 cells) at 20°C for an appropriate time, they were collected by centrifugation at 19 000× g for 15 s and immediately frozen in liquid N2. The frozen cell pellet was sonicated in 100 ml of SDS sample buffer containing 5 mM EDTA, 5 mM leupeptin, 2 mM DFP, 2 mM PMSF, 2 mM Na3VO4, 50 mM NaF and 5% 2-mercaptoethanol. The cell
G. Ishikawa et al. / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 351–357
lysate was then boiled at 90°C for 5 min. Proteins were separated by SDS – PAGE on a 7.5% gel (Laemmli, 1970) and subjected to Western blotting (Towbin et al., 1979) using anti-phosphotyrosine antibody PY20 as a primary antibody and peroxidase-conjugated anti-mouse IgG as a secondary antibody. Tyrosine phosphorylation of hemocyte proteins was measured by detection using ECL, and the bands were visualized with X-ray film.
3. Results
3.1. Effect of A74 antibody on phagocytosis by H. roretzi hemocytes It has previously been reported that the A74 antibody strongly inhibits phagocytosis of SRBCs by H. roretzi hemocytes (Takahashi et al., 1995). As shown in Fig. 1A, the A74 antibody also inhibited the phagocytosis of another target material, yeast cells, in a concentration-dependent manner. The A74 antibody showed almost 50 and 80% inhibition at concentrations of 1 mg/ml and 10 mg/ml, respectively, while control mouse IgG did not inhibit the phagocytosis. On the other hand, the A74 antibody scarcely inhibited the phagocytosis of a non-cellular target material, latex beads (Fig. 1B). It should be noted that,
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under the conditions used, 30% of total hemocytes ingested at least one yeast cell or latex bead in the absence of A74 antibody or control IgG.
3.2. Effects of kinase inhibitors on phagocytosis by H. roretzi hemocytes Next, we investigated whether protein kinase and PI3-kinase are involved in phagocytosis of yeast cells by H. roretzi hemocytes. The effects of kinase inhibitors on the phagocytosis are shown in Fig. 2. In these experiments, dimethylsulfoxide (DMSO) was used as a solvent of the inhibitors (final DMSO concentrations were 2.85 and 0.006% in Fig. 2A and B, respectively), and 29% of total hemocytes ingested at least one yeast cell in the absence or presence of 0.006% DMSO, while 27% did so in the presence of 2.85% DMSO (2.85% DMSO showed 7% inhibition against phagocytosis). As shown in Fig. 2A, the erbstatin analog and herbimycin A, tyrosine kinase inhibitors, both inhibited the phagocytosis of yeast cells. The erbstatin analog inhibited phagocytosis in a concentration-dependent manner and showed almost 90% inhibition at a concentration of 50 mM. Herbimycin A also inhibited phagocytosis in a concentration-dependent manner, but only 50% inhibition was observed even at a concentration of 50 mM. These results suggest that at least two tyrosine kinases, an erbstatin analog-sensitive one
Fig. 1. Effect of the A74 antibody on phagocytosis by H. roretzi hemocytes of yeast cells or latex beads. Phagocytosis of yeast cells (A) or latex beads (B) in the presence of the A74 antibody or control mouse IgG was measured. Approximately 200 hemocytes were inspected to check whether the hemocytes had ingested materials, and a hemocyte that had ingested at least one material was counted as a positive one. The degree of phagocytosis (%) was expressed as the ratio of the number of positive hemocytes to that of total ones. The concentrations of antibody and control IgG in B were 5 mg/ml. Bars are mean 9 S.D.; n =3.
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Fig. 2. Effects of kinase inhibitors on phagocytosis of yeast cells by H. roretzi hemocytes. The hemocytes were previously incubated for 30 min at 4°C in the presence or absence of herbimycin A (open bar in A), erbstatin analog (closed bar in A) and wortmannin (B), and yeast cells were then added to the hemocyte suspension, and the mixture was incubated at 20°C for 1 h. It should be noted that 27% of total hemocytes ingested at least one yeast cell in the absence of inhibitors but the presence of 2.85% dimethylsulfoxide (DMSO) (A), while 29% did in the presence of only 0.006% DMSO (B), as well as in the absence of DMSO. Bars in A mean9 S.D. (n =3) and the results shown in B are the means of the results of duplicate experiments.
and a herbimycin-sensitive one, are involved in phagocytosis by H. roretzi hemocytes. In the inhibition experiment with wortmannin, a specific inhibitor for PI3-kinase, the phagocytosis of yeast cells was inhibited, and 50% inhibition was observed at a concentration as low as 0.06 mM (Fig. 2B), suggesting that a PI3-kinase-like enzyme is involved in phagocytosis by H. roretzi hemocytes.
3.3. Tyrosine phosphorylation of hemocyte proteins during phagocytosis by H. roretzi hemocytes To address the question as to whether tyrosine phosphorylation of specific hemocyte proteins is involved in signal transduction during phagocytosis by H. roretzi hemocytes, we analyzed the tyrosine phosphorylation upon addition of yeast cells to the hemocytes (Fig. 3A). In a control experiment without yeast cells, H. roretzi hemocytes gradually underwent aggregation, and tyrosine phosphorylation of several proteins of 160, 110, 86, 75 and 56 kDa was observed 5 – 10 min after initiation of incubation. On the other hand, upon addition of yeast cells to the hemocytes, the above proteins of 160, 110, 86 and 75 kDa were quickly tyrosine-phosphorylated, and tyrosine phosphorylation of a new protein of 100 kDa was
Fig. 3. Tyrosine phosphorylation during phagocytosis by H. roretzi hemocytes. The hemocytes were collected in a process of phagocytosis of yeast cells and subjected to SDS – PAGE, followed by Western blotting with the anti-phosphotyrosine antibody (A). Proteins were stained with Coomassie brilliant blue (B).
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Fig. 4. Effects of the A74 antibody and kinase inhibitors on tyrosine phosphorylation during phagocytosis by H. roretzi hemocytes. After the hemocytes were pretreated with control mouse IgG (10 mg/ml, lane 2), the A74 antibody (10 mg/ml, lane 3), wortmannin (0.1 mM, lane 4), herbimycin A (1 mM, lane 5) and erbstatin analog (1 mM, lane 6), or without reagent (lane 1) at 4°C for 30 min, yeast cells were added to each hemocyte suspension and the mixture was incubated at 20°C for 10 min. The hemocytes in each case were collected and subjected to SDS – PAGE, followed by Western blotting with the anti-phosphotyrosine antibody.
also observed. In addition, it should be noted that the tyrosine phosphorylation of the A74 protein (160 kDa) was detected in both control and phagocytosis experiments. Next, we investigated whether the A74 protein, herbimycin-sensitive and erbstatin analog-sensitive tyrosine kinases, and PI3-kinase function upstream of the above tyrosine phosphorylation observed during phagocytosis. The effects of the A74 antibody, two tyrosine kinase inhibitors (herbimycin A and the erbstatin analog) and the PI3-kinase inhibitor (wortmannin) on the yeastinduced tyrosine phosphorylation of hemocyte proteins are shown in Fig. 4. Pretreatment with the A74 antibody enhanced the tyrosine phosphorylation of the A74 protein (160 kDa) and slightly enhanced the tyrosine phosphorylation of 110-, 86-, 75- and 56-kDa proteins. However, the tyrosine phosphorylation of the 100-kDa protein, which appears to be a specific event for the phagocytosis of yeast cells, was strongly inhibited by pretreatment with the A74 antibody. The three kinase inhibitors used had little effect on the tyrosine phosphorylation of hemocyte proteins, including the 100-kDa protein, at concentrations where at least 50% inhibition of phagocytosis was observed (Fig. 2). These results suggest that the A74 protein may function upstream of the tyrosine phosphorylation of the 100-kDa protein and that the two putative protein kinases and PI3-kinase may work downstream of the above tyrosine phosphorylation.
H. roretzi hemocytes undergo phagocytosis of SRBCs, yeast cells, latex beads and Escherichia coli in vitro (Ohtake et al., 1994; Takahashi et al., 1995; Azumi and Yokosawa, 1996). To determine the molecules that are involved in phagocytosis by H. roretzi hemocytes, we prepared the monoclonal antibody A74, which strongly inhibited phagocytosis of SRBCs and recognized the 160kDa antigen protein (Takahashi et al., 1995). In this study, we first showed that the A74 antibody also inhibited the phagocytosis of another cellular target material, yeast cells, together with SRBCs, but not that of a non-cellular target material, latex beads (Fig. 1). These results suggest that the A74 protein can interact with common surface materials on SRBCs and yeast cells. The A74 protein has two ITAMs, three src homology 2 (SH2) binding motifs, one src homology 3 (SH3) binding motif and two pairs of trithreonine (TTT) motifs, all of which have been proposed to function in signal transduction in mammals (Takahashi et al., 1997). We have already demonstrated that the ITAMs expressed as glutathione S-transferase-fusion proteins were tyrosine-phosphorylated by human c-Src kinase in vitro (Takahashi et al., 1997). These results led us to speculate that the ITAMs in the A74 protein are involved in the initial stage of signal transduction through tyrosine phosphorylation. To obtain in vivo evidence for the involvement of tyrosine phosphorylation in signal transduction during phagocytosis by H. roretzi hemocytes, we next investigated the effects of kinase inhibitors on phagocytosis. In mammals, the involvement of protein tyrosine kinases in FcgR-mediated signaling events has been revealed by using specific tyrosine kinase inhibitors. Several inhibitors, including herbimycin A and erbstatin, blocked phagocytosis by monocytes and macrophages and also inhibited tyrosine phosphorylation of various proteins involved in phagocytosis (Greenberg et al., 1993; Strzelecka et al., 1997). In the case of phagocytosis by ascidian hemocytes, two tyrosine kinase inhibitors (herbimycin A and the erbstatin analog) inhibited phagocytosis, but the sensitivities against the two inhibitors seemed different (Fig. 2A). Thus, it is possible that at least two different tyrosine kinases, an erbstatin analog-sensitive one and a herbimycin-sensitive one, may be involved in phagocytosis by H. roretzi hemocytes.
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In human monocytes, the involvement of PI3-kinase for FcgR-mediated phagocytosis was revealed by studies using wortmannin, a specific PI3-kinase inhibitor; the inhibitor blocked phagocytosis (Ninomiya et al., 1994). We also found that wortmannin inhibited phagocytosis of yeast cells by H. roretzi hemocytes (Fig. 2B), suggesting that a PI3-kinase-like enzyme is involved in phagocytosis by ascidian hemocytes. Next, we analyzed tyrosine phosphorylation during phagocytosis by Western blotting using an antiphosphotyrosine antibody to determine which hemocyte proteins were tyrosine phosphorylated. Immediately after the addition of target materials, the tyrosine phosphorylation of proteins of 160, 110, 86 and 75 kDa was enhanced, and a new protein of 100 kDa was tyrosine-phosphorylated (Fig. 3). Tyrosine phosphorylation of the above proteins was still observed in the presence of the three kinase inhibitors at concentrations where the inhibitors can suppress phagocytosis by 50% (Fig. 4). These results suggest that the above tyrosine phosphorylation may occur at an early stage of phagocytosis and that the two putative tyrosine kinases and the PI3-kinase-like protein may work downstream of the above event. Interestingly, we found that the A74 antibody, which can inhibit both interactions of hemocytes with conspecific hemocytes (aggregation) and with foreign materials (phagocytosis), enhanced tyrosine phosphorylation of all of the above proteins (including the A74 protein) except for the 100-kDa protein (Fig. 4). Considering the above results together with the fact that tyrosine residues in the cytoplasmic parts of FcgRs became phosphorylated upon activation of the receptors in human monocytes by their cross-linking with the specific antibodies (Duchemin et al., 1994), it can be inferred that the A74 protein functions as a signal-recognition and -transduction molecule in phagocytosis by ascidian hemocytes. With respect to the role of the 100-kDa protein in phagocytosis, the fact that the A74 antibody strongly inhibited phagocytosis-specific tyrosine phosphorylation of the 100-kDa protein (Fig. 4) led us to speculate that the 100-kDa protein, together with the A74 protein, functions at an early stage of phagocytosis, such as hemocyte recognition of foreign materials, and transduces their signals downstream through its tyrosine phosphorylation.
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