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Infection and Immunity, May 2006, p. 2876-2886, Vol. 74, No. 5
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.5.2876-2886.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Signal Transduction Mechanism Involved in Clostridium perfringens Alpha-Toxin-Induced Superoxide Anion Generation in Rabbit Neutrophils

Masataka Oda, Syusuke Ikari, Takayuki Matsuno, Yuka Morimune, Masahiro Nagahama, and Jun Sakurai^*

Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan

Received 25 September 2005/ Returned for modification 29 November 2005/ Accepted 17 February 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
Clostridium perfringens alpha-toxin induces the generation of superoxide anion (O₂^–) via production of 1,2-diacylglycerol (DG) in rabbit neutrophils. The mechanism of the generation, however, remains poorly understood. Here we report a novel mechanism for the toxin-induced production of O₂^– in rabbit neutrophils. Treatment of the cells with the toxin resulted in tyrosine phosphorylation of a protein of about 140 kDa. The protein reacted with anti-TrkA (nerve growth factor high-affinity receptor) antibody and bound nerve growth factor. Anti-TrkA antibody inhibited the production of O₂^– and binding of the toxin to the protein. The toxin induced phosphorylation of 3-phosphoinositide-dependent protein kinase 1 (PDK1). K252a, an inhibitor of TrkA receptor, and LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), reduced the toxin-induced production of O₂^– and phosphorylation of PDK1, but not the formation of DG. These inhibitors inhibited the toxin-induced phosphorylation of protein kinase C (PKC). U73122, a phospholipase C (PLC) inhibitor, and pertussis toxin inhibited the toxin-induced generation of O₂^– and formation of DG, but not the phosphorylation of PDK1. These observations show that the toxin independently induces production of DG through activation of endogenous PLC and phosphorylation of PDK1 via the TrkA receptor signaling pathway and that these events synergistically activate PKC in stimulating an increase in O₂^–. In addition, we show the participation of mitogen-activated protein kinase-associated signaling events via activation of PKC in the toxin-induced generation of O₂^–.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Clostridium perfringens alpha-toxin, which possesses lethal, hemolytic, and dermonecrotic activities and PLC and sphingomyelinase activities (37, 43, 44), has been reported to be a major pathogenic factor in the development of gas gangrene (2, 37). We have also reported that incubation of rabbit erythrocyte membranes with the toxin resulted in a biphasic production of phosphatidic acid (PA), that the initial rapid formation of PA induced by the toxin was due to activation of endogenous PLC regulated by a pertussis toxin (PT)-sensitive GTP-binding protein, and that the late formation of PA was dependent on the activation of endogenous phospholipase D (PLD) (29, 38, 39). Furthermore, we reported that the toxin activated endogenous sphingomyelinase via a PT-sensitive GTP-binding protein in sheep erythrocytes (31). Therefore, it is likely that as a first step, the toxin activates a PT-sensitive GTP-binding protein in cells. Several studies reported that alpha-toxin (34, 41) and Bacillus cereus PLC (42) activated neutrophils, measured as an increase in superoxide anion (O₂^–) (the respiratory burst) and enhancement of chemiluminescence. Grzeskowiak et al. reported that Bacillus cereus PLC induced a respiratory burst, the generation of O₂^–, and the secretion of specific granules in human neutrophils (13). Recently, we also reported that alpha-toxin stimulated adhesion to the matrix and the generation of O₂^– in rabbit neutrophils due to the formation of DG through activation of endogenous PLC by a PT-sensitive GTP-binding protein (30).

The generation of O₂^– in neutrophils has been reported to be stimulated by zymosan, 12-O-tetradecanoylphorbol 13-acetate (TPA), Ca²⁺ ionophores, and bacterial chemotatic peptides (3). The signal transduction process leading to the stimulation has been studied extensively using N-formyl-methionyl-leucyl-phenylalanine (fMLP) (18), platelet-activating factor (52), and TPA (26, 35). It has been reported that these stimuli activated mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K) in neutrophils (40, 51). Furthermore, these studies have demonstrated that the interaction of the ligands with receptors on neutrophils activates endogenous PLC with the formation of DG, which activates protein kinase C (PKC), and inositol 1,4,5-trisphosphate (IP₃), inducing the release of Ca²⁺ from the endoreticulum, and that these products act synergistically to generate O₂^–. Several studies also reported that phosphorylation of tyrosine kinases and activation of PLD were closely related to the generation of O₂^– in neutrophils stimulated with agonists (12, 22) and that activation of PLD resulted in the formation of PA, which was linked to the activation of NADPH oxidase (4, 32).

It has been reported that production of DG through activation of endogenous PLC by the toxin was closely related to the toxin-induced biological events such as hemolysis (29) and the generation of O₂^– (30), indicating that the formation of DG plays an important role in the various events induced by the toxin. However, DG itself does not have the same severe effects as the toxin. Therefore, many important questions about the mechanism of action of alpha-toxin remain unanswered. In the present study, to clarify the mechanism by which severe toxicity is induced by the toxin, we investigated the relationship between alpha-toxin-induced generation of O₂^– and signal transduction through the activation of PKC and MAPK systems in rabbit neutrophils.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Dimethyl sulfoxide (DMSO), U73122, LY294002, wortmannin, PD98059, K252a, AG1478, PP2, and 1-oleoyl 2-acetyl glycerol (OAG) were purchased from Calbiochem (San Diego, CA). Antibodies against phospho-PKC/ß, phospho-PKC, phospho-PKC/, phospho-PKCµ, phospho-PKC, phospho-TrkA, 3-phosphoinositide-dependent protein kinase 1 (PDK1), phospho-PDK1, phospho-ERK1/2, and ß-actin were from Cell-Signaling Technology (Beverly, MA). Anti-PKC antibody was obtained from BD Biosciences (San Diego, CA). Anti-TrkA antibody and normal rabbit immunoglobulin G (IgG) were obtained from Upstate (Charlottesville, VA). [-³²P]ATP (4,500 Ci/mmol) was supplied by ICN Biochemicals, Inc., Irvine, CA. Rat nerve growth factor (NGF-ß) was from R&D systems (Minneapolis, MN). All other drugs were of analytical grade.

Purification of wild-type alpha-toxin. Recombinant forms of the plasmid pHY300PLK harboring the structural genes of wild-type alpha-toxin (24) were introduced into Bacillus subtilis ISW1214 by transformation. Transformants were grown in Luria-Bertani broth containing 50 µg ampicillin/ml to an optical density at 600 nm of 0.8 to 0.85, with continuous aeration. Purification of wild-type toxin was performed as described previously (24). The purity of alpha-toxin was more than 98%.

Preparation of rabbit neutrophils. Neutrophils were purified from the peripheral blood of New Zealand White rabbits by dextran sedimentation and density gradient centrifugation and suspended at a concentration of 1 x 10⁸ cells/ml in Hanks' balanced salt solution (HBSS: 137 mM NaCl, 5.36 mM KCl, 0.337 mM Na₂HPO₄, 0.441 mM KH₂PO₄, 4.17 mM NaHCO₃, 5.55 mM glucose, pH 7.4). The purification of rabbit neutrophils was performed as described in detail previously (30). The neutrophils were routinely of high purity (>90%) and viability (>95%).

Measurement of active oxygen in rabbit neutrophils. The generation of active oxygen was evaluated by the 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo(1,2-a)pyrazin-3-one (MCLA)-dependent chemiluminescence method (25, 27). MCLA reacts with O₂^– or ¹O₂ (singlet oxygen) to emit light via the dioxetanone analogue. Rabbit neutrophils (1.0 x 10⁶ cells/ml) were incubated with alpha-toxin at 37°C in a final volume of 0.2 ml of HBSS containing 0.3 mM CaC1₂ and 1.25 µM MCLA. The chemiluminescence was measured with a chemiluminescence reader (30).

Detection of the phosphorylation of various proteins induced by alpha-toxin. Rabbit neutrophils were incubated with alpha-toxin at 37°C for 5 min in HBSS containing 0.3 mM CaCl₂, 1 mM MgCl₂, and 0.1 mM Na₃VO₄. After incubation, the reaction was terminated by the addition of 0.5 ml of ice-cold 7.5% trichloroacetic acid and kept on ice for 30 min. The precipitate was collected by centrifugation at 10,000 x g for 20 min. Phosphorylated proteins were electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to Immobilon polyvinylidene difluoride membranes (Millipore, Inc). The membranes were incubated with 5% (wt/vol) nonfat milk in TBST buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% [vol/vol] Tween 20) and probed with specific rabbit monoclonal antibodies against various phosphorylated proteins and unphosphorylated proteins (diluted 1:1,000 in TBST buffer). Detection was conducted using the enhanced chemiluminescence (ECL) kit (Amersham Bioscience). A quantitative analysis of bands was performed by densitometry (LAS-1000; Fujifilm).

Iodination of alpha-toxin. ¹²⁵I-labeled alpha-toxin was prepared according to the method of Bolton and Hunter (5). Alpha-toxin (25 µg) was incubated with 250 µCi of ¹²⁵I-labeled Bolton-Hunter reagent (2,000 Ci/mmol; Amersham Bioscience). The labeled alpha-toxin retained over 90% of its original hemolytic activity.

Immunoprecipitation of TrkA receptor. Rabbit neutrophils (1.0 x 10⁶ cells/ml) were incubated with various concentrations of ¹²⁵I-labeled alpha-toxin at 37°C for 15 min. The reaction was terminated by centrifugation, and the cells were sonicated in a lysis buffer (150 mM NaCl, 20 mM Tris [pH 7.5], 0.5% Triton X-100, 10% glycerol, 1 mM Na₂VO₄, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml pepstatin) at 4°C. Insoluble materials were removed by centrifugation at 15,000 x g for 15 min at 4°C, and supernatants were pooled and placed on ice. Pooled lysates were then immediately incubated with polyclonal antibody against a recombinant extracellular region of rat TrkA overnight at 4°C as recommended by the manufacturer (Cell Signaling Technology). Sepharose-protein G beads (Amersham Bioscience) were added to the lysates (10 µl of beads/250 µl of lysates), and the mixture was incubated for 2 h for 4°C with constant rocking. The immunoprecipitates were washed three times with the lysis buffer and boiled in SDS sample buffer containing 200 mM dithiothreitol. The proteins were resolved by 10% SDS, electrophoresed by SDS-PAGE, and transferred electrophoretically to Immobilon polyvinylidene difluoride membranes. TrkA receptor was detected with anti-TrkA receptor antibody. ¹²⁵I-labeled alpha-toxin binding to TrkA receptor was analyzed by autoradiography (FLA-2000; Fujifilm).

Measurement of diacylglycerol formation induced by alpha-toxin. Rabbit neutrophils (1.0 x10⁶ cells/ml) were incubated with alpha-toxin at 37°C in HBSS containing 0.3 mM CaCl₂, 1 mM MgCl₂, and 0.1 mM Na₃VO₄. After incubation, the reaction was terminated by the addition of chloroform-methanol (1:2 [vol/vol]), and DG was extracted and quantitated as previously described (30).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Activation of protein kinases in rabbit neutrophils treated with alpha-toxin. We reported that the toxin-induced generation of O₂^– was dependent on activation of PKC (34). It is known that phosphorylation of PKCs participates in activation of itself (33, 45). To analyze the response of PKC isoforms to alpha-toxin, the phosphorylation of PKCs in rabbit neutrophils treated with the toxin was analyzed using antibodies against various phospho-PKC isoforms (Fig. 1A). Upon exposure to 1.0 µg/ml of alpha-toxin at 37°C for various periods, phosphorylation of PKC and PKC/ reached a maximum within 30 s and later decreased in a time-dependent manner, but the toxin had no effect on the phosphorylation of PKC, PKC/ß, or PKCµ. It therefore appears that treatment of rabbit neutrophils with the toxin results in the phosphorylation of PKC and PKC /. Furthermore, the effect of calphostin C, which binds to the binding site for DG in PKC, and rottlerin, which selectivity inhibits activation of PCK and -, on the toxin-induced increase in O₂^– was investigated. As shown in Fig. 1B, the two PKC inhibitors blocked the generation of O₂^– induced by alpha-toxin in a dose-dependent manner. We also reported that alpha-toxin stimulated the generation of O₂^– in rabbit neutrophils due to the formation of DG through activation of endogenous PLC by a PT-sensitive GTP-binding protein (30). Furthermore, It is known that PKC has a binding site for DG, but PKC/ does not (21). It therefore appears that the production mechanism of O₂^– induced by alpha-toxin is closely related to activation of PKC.

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FIG.1. Phosphorylation of PKC isoforms on treatment of rabbit neutrophils with alpha-toxin. (A) Rabbit neutrophils (1.0 x 106 cells/ml) were incubated with 1.0 µg/ml alpha-toxin at 37°C for various periods, and PKC isoforms extracted by 7.5% trichloroacetic acid were subjected to SDS-PAGE and Western blotting using specific antibodies for phosphorylated PKCs. A typical result from one of five experiments is shown. (B) Rabbit neutrophils (1.0 x 106 cells/ml) were preincubated with various concentrations of rottlerin or calphostin C at 37°C for 60 min. The cells were incubated with or without 1.0 µg/ml alpha-toxin at 37°C for 15 min. Production of superoxide was monitored for 15 min based on MCLA chemiluminescence. The integrated data (area under the curve) are plotted. The generation of O2– induced by alpha-toxin alone was set as the maximal response (100%), against which all other results were compared. *, P < 0.01. Data shown are the mean ± standard error from three different experiments.

Effect of OAG on toxin-induced generation of O₂^–. We reported that treatment with the toxin or incubation with 1-oleoyl 2-acetyl glycerol (OAG) resulted in the generation of O₂^– and phosphorylation of a 40-kDa protein in rabbit neutrophils (30). As shown in Fig. 2A, the maximal level of O₂^– produced in response to 1.0 µg/ml (25 nM) of the toxin was similar to that induced by 50 µg/ml (125 µM) of OAG. Upon exposure to 25 nM of the toxin, the O₂^– level reached a maximum after about 3 min and sharply decreased. When the cells were incubated with 125 µM of OAG, O₂^– levels reached a maximum at about 12 min and remained high (Fig. 2A). The result shows that the onset of the reaction induced by the toxin is faster than that induced by OAG, and that the toxin treatment resulted in a rapid but transient increase in O₂^–. Therefore, it is likely that the formation of DG induced by the toxin is faster than the incorporation of OAG into the cells and that the DG formed in response to the toxin is rapidly metabolized. As shown in Fig. 2B, the concentration of DG produced in the cells treated with 25 nM of the toxin was about 3 µmol/1.0 x 10⁶ cells and the level of OAG incorporated by mixing 125 µM of OAG with the cells was about 120 µmol/1.0 x 10⁶ cells, indicating that the OAG level is about 40 times higher than the DG level following exposure to the toxin. On the other hand, when the cells were incubated with 5.0 µM of OAG, about 3 µmol of OAG/1.0 x 10⁶ cells was detected in the cells, but no O₂^– was observed under these conditions (Fig. 2A). To clarify the difference between the treatment with the toxin and the addition of OAG, O₂^– levels were measured in the cells treated with 2.5 nM of the toxin, which did not induce the generation of O₂^–, in the presence of 5.0 µM of OAG (Fig. 2A). As shown in Fig. 2C, the level of O₂^– produced in response to a combination of the threshold dose (2.5 nM) of the toxin and 5.0 µM of OAG was comparable to that induced by 25 nM of the toxin alone. The generation of O₂^– induced by the mixture reached a maximum after about 10 min of incubation and decreased gradually. The onset of the reaction induced by the combination was slower than that induced by 25 nM of the toxin and faster than that induced by 125 µM of OAG. These observations suggest that the toxin-induced generation of O₂^– is dependent on not only the formation of DG but also the activation of other events in the cells.

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FIG. 2. Comparison of levels of generation of O2– induced by alpha-toxin and OAG in rabbit neutrophils. (A) Rabbit neutrophils (1.0 x 106 cells/ml) were incubated with various concentrations of alpha-toxin and OAG for the indicated periods. The generation level of O2– was determined as described in Materials and Methods. Symbols: control, ; 2.5 nM alpha-toxin, ; 25 nM alpha-toxin, •; 5 µM OAG, ; 125 µM OAG, . A typical result from one of five experiments is shown. (B) Rabbit neutrophils (1.0 x 106 cells/ml) were incubated with or without alpha-toxin or OAG at 37°C for 3 or 12 min, respectively. Intracellular DG and OAG levels were determined as described in Materials and Methods. Values represent means ± standard error for five to six experiments. (C) Rabbit neutrophils (1.0 x 106 cells/ml) were incubated with or without 2.5 nM alpha-toxin in the presence of 5.0 µM OAG at 37°C for the indicated periods. Symbols: control, ; alpha-toxin plus OAG, •; alpha-toxin, ; OAG, . A typical result from one of five experiments is shown.

Involvement of PI3K and PDK1 in toxin-induced O₂^– generation. PKC is phosphorylated by PDK1 activated through PI3K linked to a TrkA (nerve growth factor high-affinity receptor) receptor that serves as a receptor for factors related to neurotrophin-3 such as NGF (17, 19). To test whether the toxin activates PDK1, rabbit neutrophils were incubated with the toxin at 37°C. The cells were subjected to SDS-PAGE, and phosphorylated proteins were analyzed by Western blotting using anti-phospho-PDK1. As shown in Fig. 3A, phosphorylation of PDK1 reached a maximum within 30 s after the incubation. In addition, the toxin dose-dependently stimulated phosphorylation of PDK1 (Fig. 3B). The increase of the protein phosphorylated by alpha-toxin treatment was not because of an increase in the protein concentration of total PDK1, as evidenced by the similar sizes of bands when blots were probed with an antibody that recognized both the phosphorylated and nonphosphorylated forms of PDK1. It therefore appears that the toxin induces phosphorylation of the PDK1, but no increase was observed for total PDK1.

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FIG. 3. Phosphorylation of PDK1 induced by alpha-toxin in rabbit neutrophils. (A) Rabbit neutrophils (1.0 x 106 cells/ml) were incubated with or without 1.0 µg/ml alpha-toxin at 37°C for the indicated periods. (B) Rabbit neutrophils (1.0 x 106 cells/ml) were incubated with or without various concentrations of alpha-toxin at 37°C for 30 s. The extracted phospho-PDK1 and PDK1 were subjected to SDS-PAGE and Western blotting using specific antibodies. A typical result from one of five experiments is shown.

Next, we examined the effect of LY294002, an inhibitor of PI3K involved in the activation of PDK1, on the toxin-induced generation of O₂^–. As shown in Fig. 4A, LY294002 at concentrations of 1.0 to 10 µM reduced the increase in O₂^– in a dose-dependent manner. Preincubation of the neutrophils with LY294002 produced a concentration-dependent inhibition of the toxin-induced phosphorylation of PDK1 and PKC (Fig. 4B). However, LY294002 did not significantly reduce the superoxide generation induced by OAG under this experimental condition (data not shown). Furthermore, 20 µM of wortmannin, an inhibitor of PI3K, also inhibited the toxin-induced generation of O₂^– and phosphorylation of PDK1 and PKC (data not shown). These inhibitors had no effect on the toxin-induced formation of DG under these conditions (Fig. 4C). U73122, an endogenous PLC inhibitor, and PT, a specific inhibitor of GTP-binding protein, inhibited the production of DG and generation of O₂^– induced by alpha-toxin, but not the phosphorylation of PDK1 (Fig. 4C and D), showing that formation of DG is independent of the phosphorylation of PDK1 in the cells treated with the toxin.

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FIG. 4. Effect of LY294002 on alpha-toxin-induced generation of O2– and phosphorylation of PKC and PDK1. (A) Rabbit neutrophils (1.0 x 106 cells/ml) were preincubated with various concentrations of LY294002 at 37°C for 60 min. The cells were incubated with or without 1.0 µg/ml alpha-toxin at 37°C for the indicated periods. Symbols: control, ; alpha-toxin, •; alpha-toxin plus 1.0 µM LY294002, ; alpha-toxin plus 5.0 µM LY294002, ; alpha-toxin plus 10 µM LY294002, . A typical result from one of five experiments is shown. (B) Rabbit neutrophils (1.0 x 106 cells/ml) treated with various concentrations of LY294002 were incubated with or without 1.0 µg/ml alpha-toxin at 37°C for 30 s. The extracted phospho-PKC and phospho-PDK1 were subjected to SDS-PAGE and Western blotting using specific antibodies. A typical result from one of five experiments is shown. (C) Rabbit neutrophils (1.0 x 106 cells/ml) were preincubated with LY294002, U73122, and pertussis toxin, and the treated cells were incubated with or without alpha-toxin at 37°C for 10 min. In the case of pertussis toxin treatment, the cells were incubated at 37°C for 120 min. Intracellular DG levels were determined as described in Materials and Methods. Values represent means ± standard error for five to six experiments. *, P < 0.01, compared with production of DG induced by alpha-toxin alone. (D) Rabbit neutrophils (1.0 x 106 cells/ml) treated with various concentrations of U73122 and pertussis toxin were incubated with or without 1.0 µg/ml alpha-toxin at 37°C for 30 s. Phospho-PDK1 was subjected to SDS-PAGE and Western blotting using specific antibodies. A typical result from one of five experiments is shown.

Several studies reported that activation of PI3K was related to the phosphorylation of TrkA receptor (28) and that generation of O₂^– was enhanced by NGF ligand to TrkA receptor (15). Next, we tested whether or not TrkA receptor exists on rabbit neutrophils. Rabbit neutrophils were solubilized in 2% SDS, electrophoresed by SDS-PAGE, and subjected to Western blotting with polyclonal antibodies against the TrkA receptor. As shown in Fig. 5A, one band at about 140 kDa reacted with anti-TrkA receptor antibody, corresponding to the molecular mass of TrkA receptor in PC12 cells. Furthermore, when the cells were incubated in the presence of 1.0 µg/ml of alpha-toxin at 37°C and phosphorylated proteins in the cells were analyzed by SDS-PAGE and Western blotting using anti-phospho-TrkA antibody, a phosphorylated protein with a molecular mass of about 140 kDa was detected (Fig. 5B). Phosphorylation of the protein reached a maximum within 30 s in the presence of the toxin (Fig. 5B). The increase in the phosphorylated protein was not due to an increase in total protein concentration of TrkA, as determined by Western blot analysis using an antibody that recognized both the phosphorylated and nonphosphorylated forms of TrkA. K252a, a TrkA receptor inhibitor, at concentrations of 1.0, 5.0, and 10 nM inhibited the toxin-induced generation of O₂^– (81, 52, and 23% of control, respectively) (Table 1) and phosphorylation of PDK1, but PP2, an inhibitor of the Src family receptor, and AG1478, an inhibitor of the endothelial growth factor receptor (EGFR), did not (1 to 10 nM for each) (Table 1 and Fig. 6A). These inhibitors had no effect on the concentration of PDK1 in the cells. It is therefore likely that the TrkA receptor is involved in the events induced by the toxin, but the Src family receptor and EGFR are not. On the other hand, K252a at a concentration of 10 nM, which decreased the level of toxin-induced phosphorylation of PDK1 by about 80%, had no effect on the toxin-induced formation of DG (Fig. 6B).

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FIG. 5. Phosphorylation of TrkA induced by alpha-toxin in rabbit neutrophils. (A) Proteins extracted from rabbit neutrophils with 7.5% trichloroacetic acid were subjected to SDS-PAGE and Western blotting using anti-TrkA antibody. Lane 1, PC12 cell lysates (positive control); lane 2, lysates of rabbit neutrophils (10 x 106 cells/ml); lane 3, lysates of rabbit neutrophils (20 x 106 cells/ml). (B) Rabbit neutrophils (10 x 106 cells/ml) were incubated with 1.0 µg/ml alpha-toxin at 37°C for various periods, and phospho-TrkA extracted from the cells with 7.5% trichloroacetic acid was subjected to SDS-PAGE and Western blotting using anti-phospho TrkA antibody. A typical result from one of five experiments is shown.

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TABLE 1. Effect of tyrosine kinase inhibitors on O2– generation induced by alpha-toxina

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FIG. 6. Effect of K252a, PP2, and AG1478 on phosphorylation of PDK1 induced by alpha-toxin in rabbit neutrophils. (A) Rabbit neutrophils (1.0 x 106 cells/ml) were preincubated with various concentrations of K252a, PP2, or AG1478 at 37°C for 60 min, and the cells were incubated with 1.0 µg/ml alpha-toxin at 37°C for 30 s. The extracted phospho-PDK1 was subjected to SDS-PAGE and Western blotting using antibodies for PDK1 and phospho-PDK1. A typical result from one of five experiments is shown. (B) Rabbit neutrophils (1.0 x 106 cells/ml) were preincubated with various concentrations of K252a, and the treated cells were incubated with or without alpha-toxin at 37°C for 10 min. Intracellular DG levels were determined as described in Materials and Methods. Values represent means ± standard error for five to six experiments.

We investigated the involvement of the TrkA receptor in the generation of O₂^– induced by alpha-toxin in rabbit neutrophils. The cells were incubated with 1.0 µg/ml of the toxin in the presence of an antibody against a recombinant extracellular region of rat TrkA at 37°C for 15 min. As shown in Fig. 7A, the antibody inhibited the generation of O₂^– induced by alpha-toxin in a dose-dependent manner, but normal rabbit IgG did not. Next, to test whether binding of the toxin to the neutrophils was inhibited by anti-TrkA receptor antibody, ¹²⁵I-alpha-toxin was incubated with rabbit neutrophils in HBSS containing various concentrations of anti-TrkA receptor antibody at 37°C for 60 s and washed with HBSS. The cells were subjected to SDS-PAGE and autoradiography. Anti-TrkA receptor antibody at concentrations of 0.1 to 1.0 µg/ml dose-dependently inhibited the binding of ¹²⁵I-alpha-toxin to the neutrophils (Fig. 7B). Normal rabbit IgG had no effect on the binding (Fig. 7B). We tested whether or not alpha-toxin directly binds to the TrkA receptor of neutrophils. The neutrophils were incubated with ¹²⁵I-alpha-toxin at 37°C for 15 min and then sonicated in the lysis buffer. The lysates were incubated with anti-TrkA receptor antibody. The immunoprecipitate was subjected to SDS-PAGE and autoradiography. As shown in Fig. 7C, ¹²⁵I-alpha-toxin was immunoprecipitated with anti-TrkA receptor antibody in a dose-dependent manner and the immunoprecipitate contained the TrkA receptor. However, ¹²⁵I-alpha-toxin and TrkA were not precipitated from the lysates using normal rabbit IgG. It therefore appears that alpha-toxin binds to TrkA receptors on the cells.

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FIG. 7. Effect of anti-TrkA antibody on alpha-toxin-induced generation of O2– and alpha-toxin binding to rabbit neutrophils. (A) Rabbit neutrophils (1.0 x 106 cells/ml) were incubated with various concentrations of TrkA antibody and 1.0 µg/ml alpha-toxin at 37°C for 15 min. Symbols: control, ; alpha-toxin, •; alpha-toxin plus 0.1 µg/ml anti-TrkA, ; alpha-toxin plus 0.5 µg/ml anti-TrkA, ; alpha-toxin plus 1.0 µg/ml anti-TrkA, ; alpha-toxin plus 1.0 µg/ml normal rabbit IgG, x. A typical result from one of five experiments is shown. (B) Rabbit neutrophils (1.0 x 106 cells/ml) were incubated with 125I-labeled alpha-toxin and various concentrations of anti-TrkA antibody or normal rabbit IgG at 37°C for 60 s. The binding of 125I-labeled alpha-toxin to the neutrophils was measured as described in Materials and Methods. A typical result from one of five experiments is shown. (C) Rabbit neutrophils (10 x 106 cells/ml) were incubated with various concentrations of 125I-labeled alpha-toxin at 37°C for 10 min. The lysated cells were immunoprecipitated using anti-TrkA antibody or normal rabbit IgG. The 125I-labeled alpha-toxin was identified by SDS-PAGE and autoradiography, and TrkA was detected by Western blotting (WB) using anti-TrkA antibody. A typical result from one of five experiments is shown.

To confirm that the TrkA receptor is involved in the toxin-evoked events, we examined the effect of NGF on the toxin-induced increase in O₂^–. Figure 8A shows that NGF, at concentrations from 10 to 50 ng/ml, which did not induce production of O₂^–, stimulated the generation of O₂^– in the presence of the threshold dose (0.1 µg/ml) of the toxin in a dose-dependent manner. On the other hand, treatment of neutrophils with 50 ng/ml of NGF alone led to the phosphorylation of PDK1 and PKC (Fig. 8C). No change was observed for total PDK1 under this condition. Furthermore, incubation of the cells with 50 ng/ml of NGF in the presence of 5.0 µM of OAG resulted in the generation of O₂^– (Fig. 8B). This is the first evidence that rabbit neutrophils have the TrkA receptor that is involved in the superoxide generation induced by alpha-toxin and NGF.

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FIG. 8. Effect of NGF on generation of O2– induced by alpha-toxin. (A) Rabbit neutrophils (1.0 x 106 cells/ml) were incubated with or without 0.1 µg/ml alpha-toxin and various concentrations of NGF at 37°C for 15 min. Symbols: control, ; alpha-toxin, •; alpha-toxin plus 10 ng/ml NGF, ; alpha-toxin plus 25 ng/ml NGF, ; alpha-toxin plus 50 ng/ml NGF, ; 50 ng/ml NGF, x. A typical result from one of five experiments is shown. (B) Rabbit neutrophils (1.0 x 106 cells/ml) were incubated with or without 5 nM OAG and 50 ng/ml NGF at 37°C for 15 min. Symbols: control, ; OAG, ; NGF, ; OAG plus NGF, (•). A typical result from one of five experiments is shown. (C) Rabbit neutrophils (1.0 x 106 cells/ml) were incubated with or without 50 ng/ml NGF at 37°C for the indicated periods. The extracted phospho-PDK1, phospho-PKC, and PDK1 were subjected to SDS-PAGE and Western blotting using specific antibodies. A typical result from one of five experiments is shown.

To clarify whether stimulation of the TrkA receptor is required for the enzyme activities of alpha-toxin, neutrophils were incubated with H148G, a variant toxin which binds to but does not hemolyse sheep and rabbit erythrocytes, at 37°C for 30 min. The neutrophils were solubilized in 2% SDS, electrophoresed by SDS-PAGE, and subjected to Western blotting with monoclonal antibodies against phospho-PDK1 and -PKC. As shown in Fig. 9, treatment of the cells with H148G resulted in the phosphorylation of PDK1 and PKC. No change was observed for total PDK1 under this condition. It therefore appears that enzyme activities of alpha-toxin are not essential for phosphorylation of PDK1 and PKC. However, H148G induced no production of DG in the cells (data not shown).

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FIG. 9. Phosphorylation of PDK1 and PKC induced by H148G in rabbit neutrophils. Rabbit neutrophils (1.0 x 106 cells/ml) were incubated with or without 1.0 µg/ml H148G at 37°C for the indicated periods. The extracted phospho-PDK1 and phospho-PKC were subjected to SDS-PAGE and Western blotting using specific antibodies. A typical result from one of five experiments is shown.

Relationship between toxin-induced O₂^– generation and MAPK system. Several studies have reported that the generation of O₂^– in neutrophils stimulated by various factors such as fMLP, TPA, and zymosan is linked to activation of the MAPK system (6, 8, 49). When rabbit neutrophils were incubated with 1.0 µg/ml of the toxin at 37°C, phosphorylation of ERK1/2 reached a maximum within 30 s and later decreased in a time-dependent manner, but not p38 MAPK and stress-activated protein kinase (SAPK)/JNK (Fig. 10A). The two bands observed for ERK1/2 represented the two isoforms: ERK1 and ERK2. Next, we examined the effect of PD98059, a MEK1/2 inhibitor, on the toxin-induced phosphorylation of ERK1/2. PD98059 at 5.0 to 10 µM inhibited the toxin-induced generation of O₂^– (data not shown) and phosphorylation of ERK1/2 (Fig. 10B). Furthermore, the phosphorylation of ERK1/2 was inhibited by U73122 and K252a (Fig. 10B). Total protein levels of ERK1/2 were unchanged under this condition. However, PD98059 did not inhibit phosphorylation of PKC (data not shown). These results suggest that PKC and MEK1/2 are located upstream of ERK1/2.

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FIG. 10. Relationship between alpha-toxin-induced generation of O2– and MAPK cascades. (A) Rabbit neutrophils (1.0 x 106 cells/ml) were incubated with or without 1.0 µg/ml alpha-toxin at 37°C for the indicated periods. The extracted phospho-MAPKs were subjected to SDS-PAGE and Western blotting using specific antibodies. *, P < 0.01, compared with phosphorylation of MAPKs induced by alpha-toxin alone. (B) Rabbit neutrophils (1.0 x 106 cells/ml) were preincubated with various concentrations of PD98059, K252a, and U73122 at 37°C for 60 min and then incubated with 1.0 µg/ml alpha-toxin at 37°C for 60 s. The extracted phospho-ERK1/2, ERK1/2, and ß-actin were subjected to SDS-PAGE and Western blotting using specific antibodies. A typical result from one of five experiments is shown.

The relationship between the toxin-induced generation of O₂^– and the p38 MAPK or SAPK/JNK system was investigated. Treatment of the cells with 1.0 µg/ml of the toxin at 37°C for 5 min resulted in no effect on the phosphorylation of p38 and SAPK/JNK (Fig. 10A). It therefore is likely that the p38 MAPK and SAPK/JNK systems play no role in the toxin-induced generation of O₂^–.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study demonstrates that alpha-toxin-induced generation of O₂^– is closely related to the activation of endogenous PKC via a combination of two events: production of DG on activation of PLC through a PT-sensitive GTP-binding protein and activation of PDK1 through the TrkA receptor.

There are three classes of PKC isotypes: classical PKC isotypes (PKC, -ß, and -) which have a C1 and C2 domain, bind DG, OAG and TPA, and are regulated by DG and Ca²⁺; novel PKC isotypes (PKC, -, -, and -), which have a C1 domain and novel C2 domain and are regulated by DG but not Ca²⁺; and atypical isotypes (/), which do not bind DG and are not regulated by these classical ligands (19). Alpha-toxin induced phosphorylation of PKC and PKC/, and the generation of O₂^– induced by the toxin was inhibited by rottlerin and calphostin C, an inhibitor of PKC. We reported that the formation of DG induced by alpha-toxin in rabbit neutrophils plays an important role in the generation of O₂^– (30). It therefore appears that the toxin-induced generation of O₂^– is dependent on the activation of PKC, through binding of PKC phosphorylated by PDK1 to DG (33, 45). PKC has been reported to play an important role in activation of the protein 1 and NF-B signaling pathway in T cells, production of inteleukin-2, and apoptosis (1, 11, 47, 48). Wang et al. reported that respiratory burst in rat neutrophils was involved in membrane-associated PKC (49). However, little is known about the function of PKC in other cells. The present study may provide clues to the role of PKC in neutrophils.

We reported that the alpha-toxin-stimulated generation of O₂^– was related to the formation of DG through activation of endogenous PLC by a PT-sensitive GTP-binding protein in rabbit neutrophils (30). In the present study, U73122, an inhibitor of endogenous PLC, blocked the toxin-induced generation of O₂^– and formation of DG in the cells, supporting that the toxin-induced increase in O₂^– is dependent on the formation of DG by endogenous PLC. However, when the level of OAG incorporated into the cells was the same as the level of DG in the cells treated with 25 nM of the toxin, the level of OAG did not induce O₂^– generation in the absence of the toxin but did in the presence of a near threshold dose (2.5 nM) of the toxin which did not induce production of DG. The result shows that the toxin-induced production of O₂^– requires not only the formation of DG, but also the activation of other events.

It has been reported that the PI3K signaling pathway has an important role in several effector functions including the generation of O₂^– (51). PI3K is known to generate phosphatidylinositol 3,4,5-trisphosphate (PIP₃), which is recognized by a pleckstrin homology domain identified as a specialized lipid-binding module (19). Several studies have reported that PDK1 requires PIP₃ as its activator for effective catalytic activity (19). Le Good et al. reported that there is a cascade involving PI3K, PDK1, and various members of the PKC superfamily in signal transduction (19). Furthermore, the function of PKC family members is reported to depend on the phosphorylation of an activation loop by PDK1 (19). LY294002 and wortmannin, both PI3K inhibitors, inhibited alpha-toxin-induced generation of O₂^– and phosphorylation of PDK1 but did not affect the toxin-induced formation of DG. The result shows that the toxin-induced activation of PI3K occurs upstream of the phosphorylation of PDK1, which is an important step in the toxin-induced generation of O₂^–. It is likely that the toxin-induced phosphorylation of PDK1 is a process independent of the toxin-induced formation of DG.

Tyrosine phosphorylation is thought to be crucial to the regulation of effector functions in neutrophils (36). It is known that stimuli that induce tyrosine kinase activity in cells evoke the generation of PIP₁, PIP₂, and PIP₃. This tyrosine kinase activity is linked to the NGF receptors with intrinsic tyrosine kinase activity. Kannan et al. reported that NGF enhances the generation of O₂^– induced by TPA in murine neutrophils (16). Ehrhard et al. reported that human monocytes express the trk proto-oncogene, encoding the signal-transducing receptor unit for NGF, and that the interaction of NGF with monocytes triggers respiratory burst activity (9). NGF, which did not induce the generation of O₂^– in rabbit neutrophils, potentiated the events triggered by the toxin and caused O₂^– to form in the presence of OAG, suggesting that a combination of the production of DG and stimulation of the NGF receptor induces severe activity in the generation of O₂^–. The TrkA receptor was detected in rabbit neutrophils and found to be phosphorylated when the cells were treated with the toxin. Furthermore, immunoprecipitation using the anti-TrkA receptor antibody revealed direct binding of the toxin to the TrkA receptor. In addition, the antibody inhibited the toxin-induced generation of O₂^–. These observations indicate that the interaction of alpha-toxin with TrkA receptors is important to the production of O₂^–. In rabbit neutrophils, K252a and LY294002 inhibited the toxin-induced generation of O₂^– and phosphorylation of PDK1 within specific concentrations, but PP2 and AG1478 did not, supporting the finding that the TrkA receptor is involved in the toxin-induced increase in O₂^–. The results obtained with the anti-TrkA antibody, LY294002, and K252a show that the activation of PI3K through direct binding of the toxin to the TrkA receptor results in production of PIP₃, which activates PDK1. In addition, PT inhibited the alpha-toxin-induced generation of O₂^– and formation of DG, but not phosphorylation of PDK1, suggesting that a PT-sensitive GTP-binding protein plays a crucial role in the coupling to endogenous PLC, but not phosphorylation of PDK1. These observations indicate that the toxin independently induces activation of both endogenous PLC via a PT-sensitive GTP-binding protein and PDK1 via the TrkA receptor.

NGF, which binds to the TrkA receptor, is reported to be required for the differentiation and survival of sympathetic and some sensory and cholinergic neuronal populations (14). Furthermore, it has been reported that NGF is involved in inflammatory responses, an increase in mast cells in neonatal rats (50), the degranulation of rat peritoneal mast cells (46), and the differentiation of specific granulocytes (16). The injection of C. perfringens cells or alpha-toxin into tissues is known to cause inflammation. Therefore, it is possible that the activation of the TrkA receptor by alpha-toxin is related to inflammation caused by C. perfringens in humans and animals.

H148G induced phosphorylation of PKC, but not production of DG, suggesting that the enzymatic activity of the toxin is essential for activation of endogenous PLC, but not activation of the TrkA receptor. It has been reported that binding of the C-domain, which does not contain the enzymatic site, to erythrocytes is important for the hemolysis induced by the toxin (23). It therefore is possible that the C-domain, the binding domain of alpha-toxin, plays a role in the binding of the toxin to the TrkA receptor and in the activation of signal transduction via TrkA receptor.

Several studies have reported that the activation of PKC by various stimuli results in the generation of O₂^– via the activation of MAPK systems (7, 8, 20, 54). K252a and U73122 inhibited the toxin-induced phosphorylation of PKC and ERK1/2 and generation of O₂^–, suggesting that the toxin-induced production of O₂^– is linked to the stimulation of the MAPK system via the activation of PKC. The toxin causes phosphorylation of ERK1/2, but not by p38 and SAPK/JNK, implying that the process is dependent on a MAPK system containing MEK1/2 and MAPK/ERK1/2, but not systems containing p38 and SAPK/JNK.

It has been reported that PA directly or indirectly activated NADPH oxidase in a cell-free system of neutrophils (10) and that PKC regulates phosphorylation of p67^phox in human monocytes (53). PKC also has been reported to activate directly NADPH oxidase (15). However, PD98059 almost completely inhibited the toxin-induced production of O₂^– near the inhibitory threshold dose of the inhibitor. Thus, it is unlikely that PA and PKC directly activate NADPH oxidase under the conditions used here.

In conclusion, we have shown that alpha-toxin induces formation of DG through the activation of endogenous PLC by a PT-sensitive GTP-binding protein and phosphorylation of PDK1 via stimulation of the TrkA receptor, so that DG and PDK1 synergistically activate PKC, and that the activation of PKC stimulates generation of O₂^– through MAPK-associated signaling events in rabbit neutrophils (Fig. 11).

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FIG. 11. Signaling events involved in alpha-toxin-activated generation of O2– in rabbit neutrophils.

 
We thank K. Kobayashi, Y. Imai, T. Taira, and Y. Saitoh for their technical assistance.

The work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan; by the Open Research Center Fund for Promotion; and by the Mutual Aid Corporation for Private School of Japan.

 
* Corresponding author. Mailing address: Faculty of Pharmaceutical Sciences, Department of Microbiology, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan. Phone: 81-088-622-9611. Fax: 81-088-655-3051. E-mail: sakurai{at}ph.bunri-u.ac.jp.

Editor: V. J. DiRita

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Infection and Immunity, May 2006, p. 2876-2886, Vol. 74, No. 5
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