Clostridium perfringens {alpha}-toxin induces rabbit neutrophil adhesion

Sadayuki Ochi1, Toshihumi Miyawaki1, Hisaaki Matsuda1, Masataka Oda1, Masahiro Nagahama1 and Jun Sakurai1

Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan1

Author for correspondence: Jun Sakurai. Tel: +81 88 622 9611. Fax: +81 88 655 3051. e-mail: sakurai{at}ph.bunri-u.ac.jp


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Clostridium perfringens {alpha}-toxin, which is one of the main agents involved in the development of gas gangrene, stimulates production in neutrophils. Exposure of rabbit neutrophils to the {alpha}-toxin induced firm adhesion of the cells to fibrinogen and fibronectin. Incubation of rabbit neutrophils and neutrophil lysates with {alpha}-toxin led to the production of diacylglycerol (DG) and L-{alpha}-phosphatidic acid (PA), respectively. The toxin-induced DG and PA formation preceded the toxin-induced adhesion of the neutrophils to fibrinogen and fibronectin, and the production of . Pertussis toxin inhibited the {alpha}-toxin-induced formation of PA, the adhesion of the neutrophils to fibrinogen and production. GTP{gamma}S stimulated the events induced by the {alpha}-toxin, whereas GDPßS inhibited them. The {alpha}-toxin stimulated phosphorylation of a protein with a molecular mass of about 40 kDa. In addition, treatment of the cells with 1-oleoyl-2-acetyl-sn-glycerol (OAG) and phorbol-12,13-dibutyrate (PDBu) stimulated cell adhesion, production of and phosphorylation of the 40 kDa protein, but had no effect on the formation of PA. The events induced by the presence of OAG and PDBu were not inhibited by pertussis toxin. Protein kinase C inhibitors, H-7, staurosporine and chelerythrine, blocked {alpha}-toxin-induced adhesion, production of and phosphorylation of the 40 kDa protein. These observations suggested that {alpha}-toxin-stimulated adhesion to the matrix and production were due to the formation of DG, through activation of phospholipid metabolism by a pertussis-toxin-sensitive GTP-binding protein, followed by activation of protein kinase C by DG.

Keywords: fibrinogen, gas gangrene

Abbreviations: DG, diacylglycerol; fMLP, formyl-Met-Leu-Pro; MCLA, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo(1,2-a)pyrazin-3-one; OAG, 1-oleoyl-2-acetyl-sn-glycerol; PA, L-{alpha}-phosphatidic acid; PDBu, phorbol-12,13-dibutyrate; PLC, phospholipase C


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Clostridium perfringens {alpha}-toxin, which possesses lethal, haemolytic, dermonecrotic and phospholipase C (PLC) activities (Titball, 1993 , 1997a , b , 1999 ; Sakurai, 1995 ), has been reported to be a major pathogenic factor in the development of gas gangrene caused by the micro-organism (Awad et al., 1995 ; Sakurai, 1995 ; Titball, 1997b ; Bryant et al., 2000a , b ). Neutrophils are known to respond to invasion by micro-organisms and to kill them through the generation of reactive oxygen metabolites. Several studies have reported that the {alpha}-toxin of C. perfringens (Patriarca et al., 1970 ; Stevens et al., 1987 ; König et al., 1997 ; Hofman et al., 2000 ), and the PLC of Bacillus cereus (Styrt et al., 1989 ), activate neutrophils, measured as an increase in formation and enhancement of chemiluminescence induced by opsonized zymosan. However, histopathology has demonstrated an absence of leukocytes within the area of necrosis associated with C. perfringens growth and the presence of these cells at the borders of the region showing necrosis (Robb-Smith, 1945 ; Stevens et al., 1997 ). Stevens et al. (1987) reported that the toxin did not alter the viability, chemotactic responsiveness or morphology of human leukocytes. Bryant & Stevens (1996) reported that exposure to {alpha}-toxin induced an up-regulation of the expression of intercellular adherence molecule (ICAM-1), E-selectin and P-selectin expression, and that exposure to {theta}-toxin induced ICAM-1 and the production of platelet-aggregating factor by endothelial cells. However, Ellemor et al. (1999) reported that an infection of C. perfringens resulted in no increase in ICAM-1. Therefore, little is known about the cause of the paucity of leukocytes within the area of necrosis caused by the micro-organism.

We have previously reported that {alpha}-toxin induces the contraction of isolated rat aorta (Fujii et al., 1986 ) and ileum (Sakurai et al., 1990 ), and that this contraction is related to the turnover of phosphatidylinositol. Notably, the toxin-induced contraction of isolated aorta was found to be closely linked to the stimulation of thromboxane A2 synthesis (Fujii & Sakurai, 1989 ). We have also reported that incubation of rabbit erythrocyte membranes with the toxin results in a biphasic production of PA and that the rapid formation of PA, induced by the toxin, is due to activation of endogenous PLC regulated by GTP-binding protein, whereas the late formation is dependent on the activation of endogenous phospholipase D (Sakurai et al., 1993 , 1994 ; Ochi et al., 1996 ). Therefore, to test if {alpha}-toxin affects the adhesion of rabbit neutrophils to tissues, we compared the effect of the toxin on the adhesion of neutrophils to matrixes. Moreover, we present evidence of a relationship between {alpha}-toxin-induced adhesion and phospholipid metabolism in rabbit neutrophils.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Purification of wild-type {alpha}-toxin and the H148G variant.
Recombinant plasmids of pHY300PLK harbouring the structural genes of wild-type {alpha}-toxin or H148G (Nagahama et al., 1995 ) were introduced into Bacillus subtilis ISW1214 by transformation. Transformants were grown in Luria–Bertani broth containing 50 µg ampicillin ml-1 to an OD600 of 0·8–0·85, with continuous aeration. The culture was centrifuged (18000 g, 20 min) and ammonium sulfate (472 g l-1) was added to the culture supernatant fluid. The ammonium sulfate fraction obtained was used as the starting material for purifying the toxins. Purification of wild-type toxin and the H148G variant was performed as described previously (Nagahama et al., 1995 ), and was followed by homogeneity-testing with SDS-PAGE and immunological techniques (Nagahama et al., 1995 ). The PLC activity of the wild-type {alpha}-toxin was determined photometrically (A405) with p-nitrophenylphosphorylcholine (Sigma) as the substrate (Kurioka & Matsuda, 1976 ). One unit of PLC activity was defined as the amount of enzyme necessary to liberate 1 µmol p-nitrophenol min-1 from p-nitrophenylphosphorylcholine at 37 °C. In this case, one unit corresponds to about 9 µg of wild-type {alpha}-toxin. H148G variant toxin bound to sheep erythrocyte membranes and phosphatidylcholine-cholesterol liposomes, but did not contain PLC (p-nitrophenylphosphorylcholine hydrolytic activity), sphingomyelinase, or haemolytic and lethal activity, as reported previously (Nagahama et al., 1995 ).

Preparation of rabbit neutrophils and cell lysates.
Rabbit blood was withdrawn from the ear arteriae of New Zealand White rabbits. Acid citrate-glucose (0·085 M sodium citrate, 0·065 M citric acid, 2% glucose) was present in each sample as an anticoagulant. Blood samples were centrifuged at 380 g for 5 min, and the supernatant containing the leukocytes was collected and mixed with 4·5% dextran (Amersham) in 0·9% NaCl. The supernatant (the plasma layer containing the leukocytes) was recovered after 30 min sedimentation and then centrifuged at 380 g for 5 min. Contaminated erythrocytes contained within the pellet were removed by haemolysis with hypotonic ammonium chloride solution (0·155 M NH4Cl, 0·119 M NaHCO3, 0·1 mM EDTA). Following centrifugation (380 g, 5 min), the white cell pellet was washed and resuspended in Hanks’ balanced salt solution without divalent cations (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 suspended cells were overlaid with lymphoprep (New England Nuclear) and centrifuged at 280 g for 20 min. The neutrophil pellet was washed and resuspended to a concentration of 1x108 cells ml-1 in HBSS. The neutrophils were routinely of high purity (>90%) and viability (>95%). Rabbit neutrophil lysates were prepared by sonication; the resulting sonicates were termed the lysate.

Measurement of active oxygen in rabbit neutrophils.
The generation of active oxygen was evaluated by the MCLA [2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo(1,2-a)pyrazin-3-one]-dependent chemiluminescence method (Nishida et al., 1989 ; Nakano, 1990 ). MCLA reacts with or 1O2 (singlet oxygen) to emit light, via the dioxetanone analogue. Superoxide dismutase (a scavenger of ), or NaN3 (a quencher of 1O2) can be used for differentiating between and 1O2-dependent luminescence. Thus, MCLA is an excellent chemiluminescence probe for the detection of in terms of its specificity and selectivity. Rabbit neutrophils (1·5x106 cells ml-1) were incubated with {alpha}-toxin at 37 °C in a final volume of 0·2 ml HBSS containing 0·3 mM CaCl2 and 1·25 µM MCLA (Tokyo Kasei Kogyo). The maximal chemiluminescence intensity was determined by measuring its peak height (c.p.m.) using a chemiluminescence reader (luminescencer-JNR; Atto, Japan). Xanthine oxidase (XO, Sigma) was assayed by the reduction of ferrocytochrome c (Sigma), in terms of the conversion of hypoxanthine (Wako Pure Chemical Industries) to urate. Briefly, the reaction mixture contained, in a final volume of 0·2 ml HBSS, 50 µM hypoxanthine and 40 µM XO. Adding the XO at 37 °C started the reaction and the change in absorbance at 550–540 nm was monitored by a spectrophotometer. In this case, one unit of XO corresponded to the generation of 0·248 nmol min-1 and a MCLA-dependent maximal chemiluminescence intensity of 744000 c.p.m.

Measurement of active oxygen in permeabilized rabbit neutrophils.
Rabbit neutrophils (2·0x106 cells ml-1) were permeabilized in HBSS containing 10 µM cytochalasin B (Nacalai Tesque) and 2 µM streptolysin O (Sigma) (preactivated by incubation with 0·45 mM dithiothreitol at 37 °C for 5 min) at 37 °C for 5 min (at this concentration of streptolysin O, 0·9±0·2% of the total lactate dehydrogenase activity was released from cells during a 30 min incubation) (Bhakdi & Tranum-Jensen, 1988 ). The permeabilized neutrophils (1·5x106 cells ml-1) were incubated with {alpha}-toxin at 37 °C in HBSS containing 0·3 mM CaCl2, 1 mM MgCl2, 0·5 mM glutathione (oxidized form) and 1·25 µM MCLA in the absence or presence of a guanine nucleotide analogue (GTP{gamma}S or GDPßS, Roche). The chemiluminescence intensity was determined by a chemiluminescence reader.

Measurement of rabbit neutrophil adhesion.
The adhesion of neutrophils to extracellular matrixes was evaluated in 24-well tissue culture plates coated with ligand. Prior to the addition of neutrophils, the plates were incubated with 500 µl per well of 50 µg human fibrinogen ml-1 (Calbiochem-Novabiochem), 50 µg bovine type I collagen ml-1 (Sigma) or 50 µg bovine fibronectin ml-1 (Nacalai Tesque) for 2 h at 37 °C. The wells were washed once with HBSS, blocked with 1% BSA in HBSS for 1 h at 37 °C, washed twice with HBSS containing 0·1% Tween 20 and then once with HBSS. Rabbit neutrophils (1·2x106 cells ml-1) were then added to individual wells. After incubation with {alpha}-toxin in the presence of 0·3 mM CaCl2 for 30 min at 37 °C, non-adherent cells were gently washed twice with warm PBS (1·06 M KH2PO4, 2·97 mM Na2HPO4 . 7 H2O, 154 mM NaCl) containing 1 mM CaCl2. Adherent neutrophils were then stained with 250 µl 0·25% rose bengal (Wako Pure Chemical Industries) solution for 10 min at room temperature. The staining solution was aspirated off and each well was washed twice with PBS and then 250 µl ethanol/PBS (1:1, v/v) solution was added. After incubation at 37 °C for 30 min, to allow the cell-retained stain to be completely dissolved, the A557 of each well was determined with a microplate spectrophotometer (SPECTRAmax 340PC, Molecular Devices), using wells containing medium alone as a control. Cell adhesion to wells coated with BSA was not detected, even when neutrophils were treated with the toxin in the presence of Ca2+.

Measurement of diacylglycerol (DG) and L-{alpha}-phosphatidic acid (PA) formation induced by {alpha}-toxin in rabbit neutrophils.
For DG measurements, rabbit neutrophils (1·5x107 cells ml-1) were incubated with {alpha}-toxin in a final volume of 0·08 ml HBSS containing 0·3 mM CaCl2 and 1 mM MgCl2 at 37 °C. After the reaction was terminated, by the addition of 0·3 ml chloroform/methanol (1:2, v/v), DG was extracted and quantified as previously described (Sakurai et al., 1993 ). In brief, DG in the extracted lipids was converted into [32P]PA by Escherichia coli DG kinase in the presence of [{gamma}-32P]ATP. [32P]PA was separated by TLC and measured in a liquid scintillation counter. The amount of DG converted to [32P]PA was calculated based upon the specific activity of [{gamma}-32P]ATP and the sample volume. A standard curve for the conversion of DG to PA was constructed using known quantities of 1-stearoyl-2-arachidonyl-sn-glycerol (Biomol Research Laboratories), and quantitated from the specific activity. A known amount of 1-stearoyl-2-arachidonyl-sn-glycerol was run with each assay to quantify the conversion.

For PA measurements, rabbit neutrophil lysates (1·5x107 cells ml-1) were incubated with {alpha}-toxin in a final volume of 0·5 ml HBSS containing 0·3 mM CaCl2, 1 mM MgCl2 and 10 µCi ml-1 (370 kBq ml-1) [{gamma}-32P]ATP (ICN Biochemicals) in the presence or absence of a guanine nucleotide analogue (GTP{gamma}S or GDPßS) at 37 °C. After the reaction was terminated by the addition of 0·5 ml ice-cold HBSS and 3·6 ml chloroform/methanol/concentrated HCl (50:100:1, by vol.), [32P]PA was extracted and quantified as previously described (Sakurai et al., 1993 ). Briefly, the extracted [32P]PA was separated by TLC and the radioactivity measured in a liquid scintillation counter.

Detection of protein phosphorylation induced by {alpha}-toxin in rabbit neutrophils.
Rabbit neutrophil lysates (1·5x107 cells ml-1) were incubated with {alpha}-toxin at 37 °C for 5 min in HBSS containing 0·3 mM CaCl2, 1 mM MgCl2, 0·1 mM Na3VO4 and 10 µCi [{gamma}-32P]ATP ml-1. After incubation, the reaction was terminated by the addition of 0·5 ml ice-cold 7·5% trichloroacetic acid and kept on ice for 30 min. The precipitate was collected by centrifugation at 10000 g for 20 min and then washed twice by centrifugation in 1 ml ice-cold 7·5% trichloroacetic acid. Phosphorylated proteins were analysed by SDS-PAGE according to Laemmli (1970) and by subsequent autoradiography.

Determination of protein concentration.
Protein concentration was determined by the Lowry method, using BSA as a standard.

Statistical analysis.
All mean values are shown with their calculated standard error (SE). Student’s t-test was used to determine the significance of differences between controls and experimental groups; a P value of 0·05 or less was considered statistically significant.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Effect of {alpha}-toxin on rabbit neutrophils
To investigate the effect of {alpha}-toxin on neutrophil adhesion to fibrinogen and on the chemiluminescence responses of rabbit neutrophils, neutrophils were incubated with various concentrations of the toxin at 37 °C for 30 min. As shown in Fig. 1(a, c), toxin added in the range 2·5–20 µg ml-1 induced adhesion of the cells to fibrinogen and chemiluminescence responses in a dose-dependent manner. The adhesion started 1–5 min after incubation when the cells were incubated with 20 µg {alpha}-toxin ml-1, and was maximal after 20 min of incubation (data not shown). Formyl-Met-Leu-Pro (fMLP), which was used as a positive control, increased cell adhesion to fibrinogen in a dose-dependent manner (Fig. 1b). The toxin-induced chemiluminescence responses began about 1·5 min after the incubation of the cells with toxin concentrations less than 20 µg ml-1, and maximal responses were observed about 4, 5 and 7 min after incubation with 20, 10 and 2·5 µg {alpha}-toxin ml-1, respectively (Fig. 1c). Toxin-induced chemiluminescence was completely inhibited by 0·5 µM superoxide dismutase, whereas it was not inhibited significantly by 0·5 mM (1O2 quencher), 20 µg catalase ml-1 (H2O2 scavenger), or 50 mM mannitol (HO· scavenger) (data not shown). Treatment of rabbit neutrophils with less than 50 µg {alpha}-toxin ml-1 caused no release of lactate dehydrogenase from the cells. Therefore, it appears that the toxin does not harm the cells, as reported by Stevens et al. (1987) . Toxin pretreated with 1 mM EDTA, and the H148G variant toxin, which does not possess PLC or haemolytic activity (Nagahama et al., 1995 ), did not induce cell adhesion to fibrinogen or a chemiluminescence response (data not shown). We tested the ability of the {alpha}-toxin-treated rabbit neutrophils to adhere to other extracellular matrix ligands, such as type I collagen and fibronectin. The toxin induced cell adhesion to these ligands in a dose-dependent manner (Fig. 1a). The adhesion of neutrophils to fibronectin was comparable with that to fibrinogen, but the toxin-induced adhesion to type I collagen was about 30% of that to fibrinogen. It is likely that the toxin-induced adhesion of rabbit neutrophils is matrix-specific.



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Fig. 1. Effect of {alpha}-toxin on neutrophil adhesion to fibrinogen and superoxide generation. (a) Rabbit neutrophils (1·2x106 ml-1) were incubated with various concentrations of {alpha}-toxin in fibrinogen- ({circ}), fibronectin- ({triangleup}) or type I collagen- ({square}) coated wells at 37 °C for 20 min. Values are means±SEfor five or six experiments. (b) Rabbit neutrophils (1·2x106 ml-1) were incubated with various concentrations of fMLP in fibrinogen-coated wells at 37 °C for 15 min. Adherent cells were determined as described in Methods. Values are means±SEfor five or six experiments. (c) Rabbit neutrophils (1·5x106 ml-1) were incubated without ({square}) or with various concentrations of {alpha}-toxin ({triangledown}, 1 µg ml-1; {blacktriangleup}, 2·5 µg ml-1; {triangleup}, 5 µg ml-1; {circ}, 10 µg ml-1; {bullet}, 20 µg ml-1) at 37 °C for different periods and the chemiluminescence was measured by a chemiluminescence reader. The results from one of five experiments yielding similar results are shown. (d) Rabbit neutrophils (1·5x106 ml-1) were incubated without ({square}) or with 10 µg wild-type {alpha}-toxin ml-1 ({circ}) or 100 µg H148G ml-1({triangleup}) at 37 °C for various periods and the chemiluminescence was measured by a chemiluminescence reader. A typical result from one of five experiments is shown.

 
Effect of {alpha}-toxin on phospholipid metabolism in rabbit neutrophils
We have previously reported that {alpha}-toxin induces haemolysis of rabbit erythrocytes through activation of phospholipid metabolism (Sakurai et al., 1993 ). Therefore, the effect of {alpha}-toxin on DG formation in rabbit neutrophils and PA formation in the cell lysates was examined. Fig. 2(a) shows that {alpha}-toxin in the range 1–20 µg {alpha}-toxin ml-1 induced formation of DG and PA in a dose-dependent manner. We then determined the time course of the {alpha}-toxin-induced production of DG and PA (Fig. 2b). The formation of DG began within 30 s of the incubation and was maximal after 1 min; formation of PA started 1 min after the incubation and gradually increased until at least 5 min. Therefore, it appears that toxin-induced DG production was followed by PA production under the conditions tested.



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Fig. 2. Effect of {alpha}-toxin on DG formation in neutrophils and PA formation in neutrophil lysates. (a) Rabbit neutrophils (1·5x107 ml-1) or lysates were incubated with various concentrations of {alpha}-toxin at 37  °C for 5 min. DG ({circ}) and PA ({bullet}) were determined as described in Methods. Values are means±SE for five or six experiments. (b) Rabbit neutrophils (1·5x107 ml-1) or lysates were incubated with 10 µg {alpha}-toxin ml-1 at 37 °C for various periods. DG ({circ}) and PA ({bullet}) were determined as described in Methods. Values are means±SE for five or six experiments. The lack of error bars indicates that the SE was smaller than the symbol.

 
Effect of pertussis toxin, GTP{gamma}S and GDPßS on the {alpha}-toxin-induced events
We have previously reported that {alpha}-toxin-induced haemolysis and PA formation in rabbit erythrocytes is due to activation of endogenous PLC, via pertussis-toxin-sensitive GTP-binding protein, which is activated by the toxin (Sakurai et al., 1993 , 1994 ; Ochi et al., 1996 ). Thus, we tested whether prior treatment of neutrophils with pertussis toxin affects {alpha}-toxin-induced adhesion and chemiluminescence responses. As shown in Fig. 3(a, b), {alpha}-toxin-induced adhesion and chemiluminescence responses in neutrophils pretreated with 1 µg pertussis toxin ml-1were about 50% as extensive as those induced by the toxin of untreated neutrophils, and the treatment with 10 µg pertussis toxin ml-1 resulted in a complete loss of the reponses induced by {alpha}-toxin. We then investigated the effect of pertussis toxin on {alpha}-toxin-induced PA formation. Fig. 4 shows that pretreatment of neutrophil lysates with pertussis toxin inhibited {alpha}-toxin-induced PA formation in a dose-dependent manner, as reported previously. To obtain further evidence for a relationship between the toxin-induced events and the GTP-binding protein, the effect of GDPßS on these toxin-induced events was examined. {alpha}-Toxin was incubated with streptolysin-O-treated neutrophils in the presence of GDPßS. The toxin-induced chemiluminescence responses in the presence of 50 and 75 µM GDPßS were about 60 and 16% of the responses seen in the absence of GDPßS, respectively (Fig. 5). Furthermore, GTP{gamma}S significantly enhanced the toxin-induced responses (data not shown). When the toxin was incubated with the lysates which had been preincubated in the presence of GTP{gamma}S or GDPßS, the formation of PA increased with increase in the dose of GTP{gamma}S and decreased with increase in the dose of GDPßS (Table 1).



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Fig. 3. Effect of pertussis toxin on {alpha}-toxin-induced neutrophil adhesion and superoxide generation. (a) Rabbit neutrophils (1·2x106 ml-1) were incubated with various concentrations of pertussis toxin at 37 °C for 120 min. Pertussis-toxin-treated neutrophils were incubated in fibrinogen-coated wells in the presence or absence of {alpha}-toxin at 37 °C for 20 min. Adherent cells were determined as described in the Methods. Values are means±SE for five or six experiments. The lack of error bars indicate that the SE was smaller than the horizontal line of the bar. *, P<0·05 compared with adhesion induced by {alpha}-toxin. (b) Rabbit neutrophils (1·5x106 ml-1) were preincubated without ({square}) or with various concentrations of pertussis toxin ({circ}, 1 µg ml-1; {triangleup}, 5 µg ml-1; {blacktriangledown}, 10 µg ml-1) at 37 °C for 120 min. Pertussis toxin-treated neutrophils were incubated without ({square}) or with 20 µg {alpha}-toxin ml-1 ({bullet}, {circ}, {triangleup}, {blacktriangledown}) at 37 °C for various periods. The results from one of five experiments yielding similar results are shown.

 


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Fig. 4. Effect of pertussis toxin on {alpha}-toxin-induced PA formation in neutrophil lysates. Rabbit neutrophils (1·5x107 ml-1) were incubated with various concentrations of pertussis toxin at 37 °C for 120 min. Pertussis-toxin-treated neutrophils were lysed and then incubated with {alpha}-toxin and 10 µCi [{gamma}-32P]ATP at 37 °C for 5 min. PA was determined as described in Methods. Values are means±SE for five or six experiments. *, P<0·05, compared with PA formation induced by {alpha}-toxin.

 


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Fig. 5. Effect of GDPßS on {alpha}-toxin-induced superoxide generation in rabbit neutrophils. Rabbit neutrophils (1·5x106 ml-1) were incubated without ({square}) or with 10 µg {alpha}-toxin ml-1({bullet}) in the presence of GDPßS ({circ}, 50 µM; {triangleup}, 75 µM; {blacktriangleup}, 100 µM) at 37 °C for various periods and the chemiluminescence was measured by a chemiluminescence reader. The results from one of five experiments yielding similar results are shown.

 

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Table 1. Effect of GTP{gamma}S or GDPßS on {alpha}-toxin-induced PA formation in permeabilized neutrophil lysates

 
Action of {alpha}-toxin on DG-activated protein kinase C
Hayakawa et al. (1986) reported that the production of the superoxide anion in human neutrophils is stimulated through phosphorylation of 44–48 kDa proteins by DG-activated protein kinase C. It also was reported that aggregation of human platelets is induced by phosphorylation of a 40 kDa protein by DG-activated protein kinase C (de Chaffoy de Courcelles et al., 1985 ; Nunn & Watson, 1987 ). We examined whether neutrophil adhesion and chemiluminescence responses were induced on addition of 1-oleoyl-2-acetyl-sn-glycerol (OAG, an analogue of DG). Both responses increased linearly with an increase in the dose of OAG and phorbol-12,13-dibutyrate (PDBu), which activate protein kinase C (data not shown). One to twenty micrograms of {alpha}-toxin was incubated with neutrophil lysates and [{gamma}-32P]ATP, and the phosphorylated proteins were analysed by SDS-PAGE. Fig. 6(a) shows that the toxin induced phosphorylation of a 40 kDa protein in neutrophils in a dose-dependent manner. When neutrophil lysates were treated with 10 µg toxin ml-1, the 40 kDa protein was strongly phosphorylated, but phosphorylation of 47, 57 and 65 kDa proteins was less than 15% of that of the 40 kDa protein. OAG and PDBu also stimulated the phosphorylation of a 40 kDa protein in the neutrophils. The effect of staurosporine and H-7, which inhibit protein kinase C, on the phosphorylation of the 40 kDa protein by the toxin was also investigated. Fig. 6(b) shows that H-7 (50 and 100 µM) and staurosporine (50 and 100 nM) inhibited phosphorylation of the 40 kDa protein by the toxin in a dose-dependent manner. Furthermore, H-7 (>50 µM) and staurosporine (20–100 nM) blocked the toxin-induced adhesion (Fig. 6c) and chemiluminescence responses in a dose-dependent manner (Fig. 6d). In addition, 25 µM chelerythrine, a protein kinase C inhibitor, significantly inhibited the toxin-induced events (data not shown). On the other hand, treatment of {alpha}-toxin with these inhibitors at 37 °C for 15 min had no effect on the PLC activity of the toxin.



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Fig. 6. {alpha}-Toxin-induced phosphorylation of 40 kDa protein and adhesion to fibrinogen. (a) Rabbit neutrophil (1·5x107 ml-1) lysates were incubated with various concentrations of {alpha}-toxin, 0·5 µM PDBu ml-1 or 10 µg OAG ml-1 at 37 °C for 5 min. Phosphorylated proteins were analysed by SDS-PAGE and autoradiography as described in Methods. The experiment was repeated with similar results. (b) Rabbit neutrophil (1·5x107ml-1) lysates were preincubated at 37 °C for 15 min with various concentrations of protein kinase C inhibitor (staurosporine or H-7) and then incubated with {alpha}-toxin and 10 µCi [{gamma}-32P]ATP ml-1 at 37 °C for 5 min. Phosphorylated proteins were analysed by SDS-PAGE and autoradiography as described in Methods. The experiment was repeated with similar results. (c) Rabbit neutrophils (1·2x106 ml-1) were preincubated with various concentrations of protein kinase C inhibitor (H-7 or staurosporine) at 37 °C for 15 min and then incubated with {alpha}-toxin in fibrinogen-coated wells at 37 °C for 20 min. Adherent cells were determined as described in Methods. Values are means±SE for five or six experiments. *, P<0·05, compared with adhesion induced by {alpha}-toxin. (d) Rabbit neutrophils (1·5x106 ml-1) were incubated without ({square}) or with 10 µg {alpha}-toxin ml-1 ({bullet}) in the presence of H-7 ({circ}, 50 µM; {triangleup}, 100 µM) or staurosporine ({blacktriangleup}, 50 nM; {blacktriangledown}, 100 nM) at 37 °C for various periods and the chemiluminescence was measured. The results from one of five experiments yielding similar results are shown.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Gas gangrene caused by C. perfringens is characterized by a rapidly spreading oedema and tissue necrosis, associated with the growth of the micro-organism (Stevens, 2000 ). Several studies have found aggregates of leukocytes in vessels at the borders of the necrotic tissue and that leukocytes were absent from the necrotic tissue (Stevens et al., 1997 ; Bryant et al., 2000a ). This phenomenon could be a result of the impairment of leukocytes by exotoxins produced by C. perfringens, or of the expression of adhesion molecules and cytokines in vivo. {theta}-Toxin is known to be cytotoxic to leukocytes (Stevens et al., 1987 ; Rossjohn et al., 1999 ), suggesting that it contributes to the paucity of leukocytes in the necrotic tissue. However, this does not explain why leukocytes accumulate in vessels at the borders of necrotic tissue.

It has been reported that {alpha}-toxin induced the expression of E-selectin and ICAM-1 in human umbilical vein endothelial cells (Bryant & Stevens, 1996 ), as well as the expression of P-selectin in vivo (Bunting et al., 1997 ; Bryant et al., 2000a ). It has also been reported that intramuscular injection of the toxin induced growth of intravascular aggregates of activated platelets, fibrin and leukocytes in a manner dependent on the fibrinogen receptor glycoprotein IIb/IIIa (gpIIb/IIIa; Bryant et al., 2000a ). However, Ellemor et al. (1999) reported that infection with C. perfringens did not induce the expression of adhesion molecules, such as ICAM-1, in vivo. In addition, it is known that selectin-dependent adhesion of leukocytes does not lead to firm adhesion unless another set of adhesion molecules is engaged (Albelda et al., 1994 ), and that the firm adhesion of neutrophils requires activation of the ß2 (CD18) integrin family, resulting in binding to one of the intercellular adhesion molecules on the surface of endothelial cells (Ley, 1996 ). If the exotoxins produced by C. perfringens activate cellular functions in neutrophils adhered to biological surfaces, neutrophils which are exposed to the toxin at the borders of the necrotic tissue would bind to adjacent vessels at the borders. In the present study, we demonstrated that treatment of neutrophils with {alpha}-toxin enhanced their adhesion to fibrinogen and fibronectin, but resulted in weak adhesion to collagen. It is known that the adhesion of neutrophils to fibrinogen is mediated by a group of ß2 and ß3 integrins (Humphries, 2000 ). Taking these findings into consideration, the toxin-induced cell adhesion suggests that the toxin induces activation of ß2 integrin(s) on neutrophils and the expression of integrin ligands, such as ICAM-1, on the surface of endothelial cells. Therefore, it is possible that the toxin-induced adhesion of the cells to matrixes promotes the growth of intravascular aggregates, as reported by Bryant et al. (2000a ).

It has been reported that {alpha}-toxin induced a respiratory burst in guinea pigs (Patriarca et al., 1970 ) and in human polymorphonuclear leukocytes (PMNLs) (Kaplan et al., 1972 ; König et al., 1997 ; Yan & Novak, 1999 ), potentiated the chemiluminescence response to opsonized zymosan in human PMNLs, and elicited superoxide production in bovine neutrophils (Styrt et al., 1989 ). {alpha}-Toxin also induced adhesion of the cells to matrix ligands, such as fibrinogen and fibronectin, and the production of reactive oxygen intermediates in rabbit neutrophils. Inhibition of MCLA chemiluminescence by superoxide dismutase, but not by , indicated that elicited MCLA luminescence without the involvement of 1O2, i.e. the production of in rabbit neutrophils. The results provide evidence of a relationship between intracellular transduction events (e.g. activation of GTP-binding protein, endogenous PLC and protein kinase C) and events such as production and adhesion of the cells to these matrix ligands in toxin-activated rabbit neutrophils. The production of DG and PA induced by the toxin followed these biological events.

Pertussis toxin inhibited toxin-induced PA formation, adhesion and production in neutrophils and GTP{gamma}S stimulated these events, but GDPßS inhibited them. These observations suggest that the activation of phospholipid metabolism plays an important role in these events, which are induced by the toxin, and is dependent on activation of the pertussis-toxin-sensitive GTP-binding protein. Ohta et al. (1985) reported that fMLP induced production of the superoxide anion which was mediated by the pertussis-toxin-sensitive GTP-binding protein present in human neutrophils. In addition, we have reported that {alpha}-toxin induced the metabolism of phospholipid and haemolysis through activation of the pertussis toxin-sensitive GTP-binding protein in rabbit erythrocytes (Sakurai et al., 1994 ; Ochi et al., 1996 ). The process of toxin-induced neutrophil activation is supported by the results obtained with fMLP and by the haemolysis induced by the toxin.

{alpha}-Toxin stimulated the production of DG and PA with phosphorylation of a 40 kDa protein in rabbit neutrophils. However, OAG and PDBu, both protein kinase C activators, activated cell adhesion and production, but did not enhance DG and PA production. The results show that the phosphorylation of the 40 kDa protein, induced by these activators, occurred downstream of the toxin-induced phospholipid metabolism. In addition, protein kinase C inhibitors inhibited the toxin-induced production, cell adhesion and phosphorylation of the 40 kDa protein. It appears that the toxin-induced adhesion is closely related to the production of DG, a protein kinase activator, through activation of phospholipid metabolism. Several studies have shown that {alpha}-toxin can elicit the activation of protein kinase C in rat skeletal muscle (Henriksen et al., 1989 ) and mouse epidermal HEL-37 cells (Jones & Murray, 1986 ). Grzeskowiak et al. (1985) reported that the neutrophil responses induced by the toxin, activation of a respiratory burst and secretion of specific granules, were dependent on the formation of DG with the breakdown of phospholipids. They hypothesized the involvement in the neutrophil responses of protein kinase C. Our result was consistent with the hypothesis proposed by Grzeskowiak et al. (1985) . Yan & Novak (1999) reported that treatment of human neutrophils with tumour necrosis factor {alpha} and fMLP, which stimulated adhesion of the cells to fibrinogen, resulted in the phosphorylation of cellular proteins with molecular masses of approximately 115 kDa. Hayakawa et al. (1986) reported that treatment of human neutrophils with DG caused the production of the superoxide anion and the phosphorylation of proteins with molecular masses of 44–48 kDa. Fuchs et al. (1997) reported that phorbol ester induced phosphorylation of a 40 kDa protein in HL60 cells that had differentiated into neutrophils, and that the protein phosphorylation was closely related to the kinetics of production. Therefore, the toxin-induced adhesion and production appears to be involved in the phosphorylation of cellular proteins in neutrophils. The phosphorylated 40 kDa protein was reported to be p40phox, a soluble component of NADPH oxidase (Fuchs et al., 1997 ; Babior, 1999 ). However, Bianca et al. (1999) reported that antigen–antibody reactions of antibodies against the components of NADPH oxidase were species-specific and identified human and mouse NADPH oxidase components. Rabbit NADPH oxidase components, however, have not yet been isolated. Therefore, in the present study, the phosphorylated 40 kDa protein in rabbit neutrophils treated with the toxin was difficult to identify.


   ACKNOWLEDGEMENTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
This research was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We thank K. Kobayashi and S. Ikari for their technical assistance.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 21 March 2001; revised 23 July 2001; accepted 20 September 2001.