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Botulinum C2 Toxin ADP-Ribosylates Actin and Enhances O2- Production and Secretion but Inhibits Migration of Activated Human Neutrophils

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Botulinum C2 Toxin ADP-Ribosylates Actin and Enhances O2- Production and Secretion but Inhibits Migration of Activated Human Neutrophils Botulinum C2 toxin ADP-ribosylates actin and enhances O2- production and secretion but inhibits migration of activated human neutrophils. J Norgauer, … , R Seifert, K Aktories J Clin Invest. 1988;82(4):1376-1382. https://doi.org/10.1172/JCI113741. Research Article The binary botulinum C2 toxin ADP-ribosylated the actin of human neutrophils. Treatment of human neutrophils with botulinum C2 toxin for 45 min increased FMLP-stimulated superoxide anion (O2-) production 1.5-5-fold, whereas only a minor fraction of the cellular actin pool (approximately 20%) was ADP-ribosylated. Effects of botulinum C2 toxin depended on toxin concentrations, presence of both components of the toxin, and incubation time. Cytochalasin B similarly enhanced O2- production. The effects of botulinum C2 toxin and cytochalasin B were additive at submaximally, but not maximally effective concentrations and incubation time of either toxin. Botulinum C2 toxin also enhanced stimulation of O2- production by Con A and platelet-activating factor, but not by phorbol 12-myristate 13-acetate (PMA). Botulinum C2 toxin increased FMLP-induced release of N-acetyl-glucosaminidase by 100-250%; release of vitamin B12- binding protein induced by FMLP and PMA was enhanced by approximately 150 and 50%, respectively. Botulinum C2 toxin blocked both random migration of neutrophils and migration induced by FMLP, complement C5a, leukotriene B4, and a novel monocyte-derived chemotactic agent. The data suggest that botulinum C2 toxin-catalyzed ADP-ribosylation of a minor actin pool has a pronounced effect on the activation of human neutrophils by various stimulants. Find the latest version: https://jci.me/113741/pdf Botulinum C2 Toxin ADP-Ribosylates Actin and Enhances 02 Production and Secretion but Inhibits Migration of Activated Human Neutrophils Johannes Norgauer,* Eckhard Kownatzki,* Roland Seifert,l and Klaus Aktories* *Rudolf-Buchheim-Institutfur Pharmakologie der Justus-Liebig-Universitdt, D-6300 Giessen, Federal Republic ofGermany; tAbteilung Experimentelle Dermatologie der Universitdts-Hautklinik, D-7800 Freiburg i.B., Federal Republic of Germany; and §Institutfur Pharmakologie der Freien Universitdt Berlin, D-1000 Berlin, Federal Republic of Germany Abstract terfere with leukocyte activation caused by various chemotac- tic stimulants (7-9). Not only phenomena obviously related to The binary botulinum C2 toxin ADP-ribosylated the actin of cell motile functions are regulated by cytoskeletal elements. human neutrophils. Treatment of human neutrophils with bot- Several findings indicate an involvement of cytoskeleton pro- ulinum C2 toxin for 45 min increased FMLP-stimulated su- teins in activation of NADPH oxidase by chemotactic agents peroxide anion (O°) production 1.5-5-fold, whereas only a (7-10). Again, this conclusion was mainly drawn from the minor fraction of the cellular actin pool (- 20%) was ADP-ri- findings that cytochalasins drastically increased the O2 pro- bosylated. Effects of botulinum C2 toxin depended on toxin duction caused by neutrophil stimulatory agents like FMLP, concentrations, presence of both components of the toxin, and platelet-activating factor (PAF),' and Con A (7-9). incubation time. Cytochalasin B similarly enhanced O2 pro- Botulinum C2 toxin belongs to a class of bacterial ADP-ri- duction. The effects of botulinum C2 toxin and cytochalasin B bosyltransferases that modify actin (1 1, 12). The toxin is bi- were additive at submaximally, but not maximally effective nary in structure and consists of components I (C2 I) and II concentrations and incubation time of either toxin. Botulinum (C2 II) (13). Whereas C2 I (Mr 50,000) possesses ADP-ribosyl- C2 toxin also enhanced stimulation of O2 production by Con A transferase activity, C2 II (Mr 100,000) is apparently involved and platelet-activating factor, but not by phorbol 12-myristate in the binding of the toxin to the cell membrane. The substrate 13-acetate (PMA). Botulinum C2 toxin increased FMLP-in- of botulinum C2 toxin is nonmuscle G actin, but not polymer- duced release of N-acetyl-glucosaminidase by 100-250%; re- ized F actin (12, 14). Moreover, the covalent modification of lease of vitamin B12-binding protein induced by FMLP and actin blocks the ability of actin to polymerize (1 1, 12, 15). This PMA was enhanced by - 150 and 50%, respectively. Botuli- ability to inhibit actin polymerization thus qualifies botuli- num C2 toxin blocked both random migration of neutrophils num C2 toxin as a new tool with which to study the role of and migration induced by FMLP, complement C5a, leuko- actin in various cell functions. Therefore, these results triene B4, and a novel monocyte-derived chemotactic agent. prompted us to study the effects of botulinum C2 toxin on The data suggest that botulinum C2 toxin-catalyzed ADP-ri- activation of human neutrophils by various stimulants and bosylation of a minor actin pool has a pronounced effect on the compare its effects with those of cytochalasin B. activation of human neutrophils by various stimulants. Methods Introduction Materials. FMLP, SOD, cytochalasin B, l-O-alkyl-2-acetyl-sn-gly- Activation of neutrophils by chemotactic agents induces cellu- cero-3-phosphocholine (PAF), phorbol 12-myristate 13-acetate lar responses like cell shape change, migration, degranulation, (PMA), thymidine, leupeptin, p-nitro-phenyl-N-acetyl-i-glucosamide, and phagocytosis (1). All these events depend on cellular mo- and PMSF were obtained from Sigma Chemical (Deisenhofen, FRG). tile functions and the dynamic processes involved in restruc- Dextran T 500 and Ficoll-Hypaque were purchased from Pharmacia turing of the cytoskeleton (1, 2). Recent studies have indicated Fine Chemicals (Freiburg, FRG); ATP from Boehringer Mannheim- GmbH (Mannheim, FRG); and Triton X-100 from SERVA Feinbio- that actin, besides its role in muscle contraction, is basically chemical & Co. (Heidelberg, FRG). Xylol and toluene were obtained involved in cellular motile processes of such nonmuscle cells from Roth GmbH & Co. KG (Karlsruhe, FRG); [57Co]- as neutrophils and platelets (2, 3). In nonmuscle cells, these cyanocobalamine and [32P]NAD from Amersham Buchler (Braunsch- motile functions are apparently associated with changes in the weig, FRG); and Con A from Miles Laboratories, Inc. (Munich, FRG). state of actin polymerization. It thus has been shown that Leukotriene B4 (LTB4), the chemotactic split peptide C5a, and the activation of neutrophils or platelets is accompanied by a rapid monocyte-derived chemotactic agent (MOC) were produced as de- increase in polymerized actin (3-5). Moreover, agents that scribed (16). Botulinum C2 toxin was prepared and activated essen- block actin polymerization, like cytochalasins (6), largely in- tially as described (13). Pertussis toxin was kindly donated by Dr. Yajima (Kobe, Japan). 3-,Am-pore size cellulose nitrate filters were purchased from Sartorius GmbH (Gottingen, FRG). Address reprint requests to Dr. Klaus Aktories, Rudolf-Buchheim- Preparation of human neutrophils. To determine °2 production Institut fir Pharmakologie, Frankfurterstrasse 107, D-6300 Giessen, and for the ADP-ribosylation assay, human neutrophils were isolated Federal Republic of Germany. from freshly drawn venous blood obtained from healthy donors, es- Received for publication 9 February 1988 and in revised form 20 sentially as described by Markert et al. (17). To determine migration May 1988. J. Clin. Invest. 1. Abbreviations used in this paper: C2 I, component I ofbotulinum C2 © The American Society for Clinical Investigation, Inc. toxin; C2 II, component II of botulinum C2 toxin; MOC, monocyte- 0021-9738/88/10/1376/07 $2.00 derived chemotactic agent; PAF, platelet-activating factor (l-O-alkyl- Volume 82, October 1988, 1376-1382 2-acetyl-sn-glycero-3-phosphocholine). 1376 J. Norgauer, E. Kownatzki, R. Seifert, and K. Aktories and enzyme release, neutrophils were isolated as described (16). Either Figure 1. FMLP con- isolation procedure resulted in 95% of neutrophils with viability 24 centration dependence > 95%, as measured by trypan blue exclusion. Toxin treatment of 20 of O° production by neutrophils for up to at least 150 min did not reduce viability of cells. 16 human neutrophils. ADP-ribosylation assay. ADP-ribosylation was performed essen- L_ CX 12/ (Top) Influence of cyto- tially as described (12, 15). Briefly, neutrophils (10' cells/ml) were > 8 { /chalasin B. Human incubated I E / /neutrophils (106 cells) with 400 ng/ml C2 and 1.6 Mg/ml C2 II of botulinum toxin 4. in a buffer containing 138 mM NaCl, 6 mM KCI, 2 mM CaCl2, 1 mM were preincubated with Na2HPO4, 5 mM NaHCO3, 5.5 mM glucose, and 20 mM Hepes, pH , I , , , , (o) and without (X) 2 7.5, for the indicated periods of time. Thereafter, the cells were washed 0 10-9 I -8 1 -7 10-6 ug/ml cytochalasin B twice; lysed in a medium containing 10 mM triethanolamine-HCI (pH fMLP (M) for 2 min. Treated neu- 7.5), 0.5 mM PMSF, 0.1 g/ml leupeptin, and 25 mM EDTA; frozen; trophils were stimulated thawed; homogenized; and finally used in the ADP-ribosylation assay. with the indicated con- ADP-ribosylation of neutrophil lysates (- 1 mg of protein/tube) was 24 centrations of FMLP carried out in a buffer containing 50 mM triethanolamine-HCl (pH C 20i for 15 minandthereaf- 7.4), 10 mM thymidine, 0.5 mM ATP, 4 mM MgCl2, 1 MM [32P]NAD 16/ terO production was (- I uCi), 0.02% BSA (wt/vol) and 1 Mg/mi botulinum C2 toxin com- 2M 12 determined asde- O.,,, ponent I. The incubation was carried out for 10 min at 37°C. The o8 Xscribed. (Bottom) Influ- reaction was stopped by the addition of 20 gl of a buffer containing d" E ence of botulinum C2 10% (wt/vol) SDS, 10% (wt/vol) saccharose, 1% (vol/vol) 2-mercap- *5 toxin. Human neutro- toethanol, and 100 mM Tris-HCI, pH 7.4. Thereafter, the radioactively o * _'__ phils (10' cells) were labeled proteins were analyzed by SDS-PAGE according to Laemmli 0 10-9 10-8 10-7 10-6 preincubated with (o) (18).
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