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
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ABSTRACT |
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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--phosphatidic acid; PDBu, phorbol-12,13-dibutyrate; PLC, phospholipase C
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INTRODUCTION |
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We have previously reported that -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
-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
-toxin-induced adhesion and phospholipid metabolism in rabbit neutrophils.
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METHODS |
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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Rabbit blood was withdrawn from the ear arteriae of New Zealand White rabbits.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
-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 550540 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
-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
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 -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--phosphatidic acid (PA) formation induced by
-toxin in rabbit neutrophils.
For DG measurements, rabbit neutrophils (1·5x107 cells ml-1) were incubated with -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 [
-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 [
-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 -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) [
-32P]ATP (ICN Biochemicals) in the presence or absence of a guanine nucleotide analogue (GTP
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 -toxin in rabbit neutrophils.
Rabbit neutrophil lysates (1·5x107 cells ml-1) were incubated with -toxin at 37 °C for 5 min in HBSS containing 0·3 mM CaCl2, 1 mM MgCl2, 0·1 mM Na3VO4 and 10 µCi [
-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). Students 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.
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RESULTS |
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DISCUSSION |
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It has been reported that -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
-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 -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
).
-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
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
-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.
-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
-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
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 4448 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 antigenantibody 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.
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ACKNOWLEDGEMENTS |
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Received 21 March 2001;
revised 23 July 2001;
accepted 20 September 2001.