INFECTION AND IMMUNITY, July 1979, p. 187-190 0019-9567/79/07-0187/04$02.00/0

Vol. 25, No. 1

Inhibition by Cholera Toxin of Rat Polymorphonuclear Leukocyte Chemotaxis Demonstrated In Vitro and In Vivo MONIQUE ROCH-ARVEILLER,' PATRICE BOQUET,2 DAVID BRADSHAW,' AND JEAN-PAUL

GIROUD`* Department of Pharmacology, H6pital Cochin, 75014 Paris,1 and Institut Pasteur, Unite des Antigenes Bacteriens, 75015 Paris, France2 Received for publication 2 April 1979

The effect of cholera toxin on the chemotaxis of rat polymorphonuclear leukocytes (PMN) was studied using a technique in which the movement of the cells towards a laser-lysed erythrocyte is followed under a phase-contrast microscope. In vitro studies indicated that the intact toxin was capable of inhibiting PMN chemotaxis in a dose-dependent manner at doses ranging from 1 to 100 ng/ ml. Subunits A and B of the toxin were without inhibitory activity when used alone, but after recombination their ability to inhibit chemotaxis was similar to that of the intact toxin, suggesting that the toxin is acting intracellularly. Cholera toxin has been reported to act in other systems via stimulation of adenyl cyclase with consequent elevation of intracellular cyclic adenosine 5'-monophosphate (cAMP) levels. It appears that this mechanism may also account for its ability to inhibit chemotaxis since there was a correlation, at all doses tested, between inhibition of chemotaxis and increased intracellular cAMP levels. Cholera toxin was also found to be active in vivo in that, after intrapleural injection of the toxin, the chemotaxis of cells subsequently recovered from the pleural cavity was markedly reduced. These results support previous findings which suggest that modification of leukocyte cAMP levels can influence the chemotactic responsiveness of these cells.

Chemotaxis is probably the main mechanism by which polymorphonuclear leukocytes (PMN) move towards sites of infection, tissue damage, or both. Various groups of workers have obtained evidence suggesting that cyclic nucleotides may play an important role in inflammatory processes, and a correlation has been suggested between cyclic nucleotide levels and the degree of PMN chemotaxis (5, 7, 13, 16-18). So far these activities have been demonstrated using either cyclic nucleotide derivatives or various drugs which, among their pharmacological properties, activate adenylate cyclase or inhibit phosphodiesterase. It was thus of interest to investigate the activity of a molecule which raises specifically and for a long period the intracellular level of cyclic adenosine 5'-monophosphate (cAMP). Cholera toxin in this respect appears to be useful (3, 19). The exo-enterotoxin of Vibrio cholerae (84,000 molecular weight) is composed of two subunits, A and B. Subunit A contains two peptides, Al and A2, linked by a disulfide bridge (9, 10), and this fragment has been shown to promote the intracellular activation of adenylate cyclase (12). The B subunit binds ganglioside GM, present in eucaryotic cell membranes. This

fragment is responsible for the cell membrane attachment of the whole toxin and probably mediates the passage of the A fragment to the cytoplasm through the lipid bilayer (11, 20). In the present work we used cholera toxin to raise the cAMP level in rat PMN, and the effect on the chemotaxis of these cells was studied. MATERIALS AND METHODS Preparation of normal isologous serum. Inbred pathogen-free rats were exsanguinated, and the blood was centrifuged at 500 x g for 5 min. The supernatant was recentrifuged at 4,000 x g for 10 min, and the resulting supernatant was used as normal serum. Collection of leukocytes. PMN were harvested from the pleural cavity 4 h after the intrapleural injection of 1 ml of isologous serum (14). In vitro studies. For in vitro studies, approximately 1 pl of whole blood was added to the PMN preparation to provide a few erythrocytes. The cells were then washed three times using an enriched Eagle type medium (NCTC 135) (Flow Laboratories, Asnieres, France) and then incubated in cholera toxin for 30 min at 37°C. After incubation the solution was centrifuged for 5 min at 500 x g, and the cells were

washed once and then resuspended in a small volume of the same medium. The chemotaxis of the cells was then assessed as described below.




In vivo studies. For in vivo studies a control population of cells was withdrawn from the pleural cavity of rats injected 3.5 h earlier with isologous serum. Cholera toxin was then injected in a volume equivalent to that withdrawn (0.4 ml). After 30 min, cells were again withdrawn, 1 Md of whole blood was added, and after washing once the chemotaxis of the cells was assessed and compared with controls. Assessment of chemotaxis. Chemotaxis was assessed by the method used initially by Bessis and Burt6 (1) and developed by Giroud et al. (14) in which the chemotactic movement of PMN towards an erythrocyte lysed by a microbeam laser is followed under a phase-contrast microscope. To quantify the chemotactic response, three parameters were measured, namely, the number of cells in the microscope field at t = 0 (designated A), the number of cells in the field at t + 10 min after lysis of the red cell, and the number of cells attached to the target cell at t + 10 min (designated B). For a comparison between test and control values, the ratio (B/A) x 100 was calculated and used for statistical analysis using a paired t test. The difference in this ratio between test and control values quantifies the effect of the test agent and is negative for inhibition of chemotaxis and positive for stimulation of chemotaxis. Extraction and determination of cAMP from leukocytes. Five rats were injected intrapleurally with 1 ml of isologous serum, and 4 h later the fluid was withdrawn from the pleural cavity. Its cellular content consisted of approximately 95% PMN. The fluid was centrifuged for 5 min at 500 x g, and the supernatant was discarded. The cells were then washed twice with medium 199 (Flow Laboratories, Asnieres, France) and suspended in 7.5 ml of the same medium, and the number of cells was determined using a Hycel counter. A 0.5-ml volume of the cell suspension was then taken and incubated in cholera toxin for 30 min at 370C. Triplicate incubations were performed for each of four doses of toxin (0.1, 1, 10, and 100 ng/ ml) plus control incubations in medium 199. After discarding the supernatant, approximately 0.5 ml of a solution containing equal volumes of medium 199 and absolute alcohol was added, and the solution was sonicated twice for 15 s with a 15-s interval between the two sonications. After centrifuging for 10 min at 4,800 X g, the supernatant was evaporated to dryness using a Rotavapor (Buchi) (30 rpm, 37°C), and a cAMP determination was performed on the residue by the method of Brown et al. (2). cAMP levels were then expressed as picomoles per 108 cells. Source of cholera toxin. Purified cholera toxin was purchased from Schwartz/Mann. The protein, at a concentration of 2 mg/ml in tris(hydroxy-


acid-azide-NaCl buffer (pH 7.5) was stored frozen in small aliquots. The A and B subunits of the toxin were purified by the method of Finkelstein et al. (9). Figure 1 shows the sodium dodecyl sulfate gel electrophoresis patterns of cholera toxin and its fragments.

RESULTS Effect of cholera toxin on PMN chemotaxis. Isolated PMN were incubated in vitro for


.. 0X


FIG. 1. Electrophoresis pattern of cholera toxin before and after separation into its component subunits A and B. Gel electrophoresis was performed with sodium dodecyl sulfate gels containing 15%polyacrylamide using a buffer containing 0.025 M tris(hydroxymethyl)aminomethane, 0.192 M glycine, and 0.1% sodium dodecyl sulfate.

30 min with various concentrations of cholera toxin and then studied for their speed and direction of movement towards a target as described in Materials and Methods. A marked decrease of the cell chemotaxis was observed, compared to control preparations, for doses of toxin ranging from 1 to 100 ng/ml (Table 1). With a dose of 0.1 ng of toxin per ml, no significant modification could be detected. The speed of the cells was calculated from a measurement of the distance they migrated during a 3-min period and averaged 12 ,um/min. None of the doses of cholera toxin used in this study produced any significant variation in this speed. To rule out the possibility that the toxin B subunit could induce the modification of chemotaxis by simply binding to the membrane ganglioside GM,, the effect of the A and B subunits used separately was assessed. Neither the A subunit nor the B subunit had any significant effect on PMN chemotaxis when used alone. However, when the two subunits were recombined, the inhibitory effect of the native toxin was recovered (Table 2). A similar decrease in cell chemotaxis could be induced in vivo by injecting the toxin directly into the pleural cavity of rats as described in Materials and Methods (Table 3). Effect of cholera toxin on leukocyte cAMP levels. As shown in Fig. 2, leukocytes

VOL. 25, 1979


TABLE 1. Effect of cholera toxin on PMN chemotaxis in vitro B A No. of PMN in the

Dose (ng/ml)

No. of PMN in the field at t = 0 Control

100 10 1 0.1 aP < 0.001. b P < 0.05.

252 240 189 93

Treated 214 178

175 172

field at in+ 10

Control 467

415 421 168


No. of




on the




Control Treated 56 21



301 250

140 135

45 45

317 290

139 57

84 80


No. of PMN

Ratio B/A (xlOO)


TABLE 2. In vitro effect of toxin subunits (isolated or associated) on PMN chemotaxis

No. of PMN Frcton MnteIn the at field at field lt t+10

No. of PMN on the target at t + 10 min



to con-


A Control Treated

183 215

384 424

131 142

72 66


Control Treated

202 254

440 544

153 173

76 68



A+B 117 85 309 Control 138 39 -46 286 69 Treated 152 100 A and B were used at individually aFractions ng/ml. The mixture A + B contained 50 ng of fraction A and 50 ng of fraction B, each per ml.

bp < 0.001.

incubated in cholera toxin showed a dose-dependent increase in intracellular cAMP levels such that the cAMP levels of leukocytes incubated in 100 ng of cholera toxin per ml was more than twofold greater than control levels. The increase in cAMP levels correlated well with the inhibition of chemotaxis produced by the same doses of the toxin (Fig. 2).

DISCUSSION Our results indicate that cholera toxin is capable of inhibiting rat PMN chemotaxis after either in vivo or in vitro administration. Previous workers (9, 10) demonstrated that the activity of the toxin is dependent on the presence of both types of subunit of which it is composed, the B subunit being necessary for the binding of the toxin to the cell membrane so that the active A subunit can enter the cell and exert its effect (4, 11, 20). The present results are consistent with these findings since the subunits A or B used alone showed no effect on PMN chemotaxis

56 74 61

25 48 47


Ratio difference, treated to control

-35a -31a -26 -14

whereas recombination of the two resulted in an inhibition of chemotaxis equal to that obtained with the intact toxin. As with the other biological properties of cholera toxin, such as its stimulatory effects on membrane ion exchange (8), lipolysis in fat cells (15), and glycogenolysis in liver cells and platelets (22), the inhibition of chemotaxis by the substance appears to be related to an increased intracellular cAMP level produced by stimulation of adenyl cyclase. Thus, incubation of leukocytes in varying doses of toxin resulted in a dose-related increase in cAMP levels which correlated well with the observed inhibition of chemotaxis. We have previously demonstrated that the intrapleural administration of dibutyryl cAMP in rats inhibited PMN chemotaxis (13). However, diffusion of the compound within the animal, its possible inactivation, and the possible delay or difficulty of its entry into leukocytes necessitated the use of a high dose of the compound so that inhibition of chemotaxis by nonspecific effects cannot be rule out. By using cholera toxin, we have been able to show a direct correlation between chemotaxis inhibition and increased intracellular cAMP levels. Rivkin et al. (18) have shown that cholera toxin is capable of raising cAMP levels in rabbit peritoneal neutrophils and of inhibiting the chemotaxis of these cells. However, the correlation between the doses required to produce these effects was not altogether consistent since, for example, a dose of toxin of 10 ng/ml which raised cAMP to the same levels as did 50 ng/ml had no effect on chemotaxis, whereas the latter dose produced a 27% inhibition. These workers also observed an inhibitory effect of the toxin on the spontaneous motility of the rabbit neutrophils. Our results indicate that, by contrast, cholera toxin affects only the direction of movement of rat PMN, without influencing their speed either before or after their response to the chemotactic stimulus. Whether the discrepancy between the two series of results is due to species or methodological differences remains to be determined.



ROCH-ARVEILLER ET AL. TABLE 3. Effect of cholera toxin on PMN chemotaxis in vivo

A No.field of PMN Dose at t =in0the (ng/ml) (ng/ml) Control Treated

166 181

100 1

159 188

No. of PMNminthe field at th+10

B of PMN at t +on10the

Ratio B/A


















Ratiotreated difference,

control ~~~~~~~~~~~~~~~~~~~~~~~

Control Treated



ap< 0.01. P < 0.05.

50~~~~~~~~~~~~~~~ 0









Cholera toxin concentration (ng/mb

FIG. 2. cAMP determinations and assessment of chemotaxis were performed as described in the text and are expressed as described therein. For chemotaxis, the scale for the "ratio difference" is the same as that for the concentration of cAMP.

That variations in cAMP levels can influence inflammatory reactions is well known (6, 21), and our results lend further support to the concept that this influence may be exerted, at least in part, via effects on leukocyte migration. ACKNOWLEDGMENTS We gratefully acknowledge the expert technical assistance of Olivier Muntaner and the financial support given by Institut National de la Sante et de la Recherche M6dicale.

LITERATURE CITED 1. Bessis, M., and B. Burte. 1964. Chimiotactisme apres destruction d'une cellule par microfaisceau laser. C. R. Soc. Biol. 158:1995-1997. 2. Brown, B. L., R. P. Ekins, and J. D. M. Albano. 1972. Saturation assay for cyclic AMP using endogenous binding protein, p. 25. In P. Greengard and G. A. Robison (ed.), Advances in cyclic nucleotide research, vol. 2. Raven Press, New York. 3. Carpenter, C. C. J., R. B. Sack, J. C. Feeley, and R. W. Steenberg. 1968. Site and characteristics of electrolyte loss and effect of intraluminal glucose in experimental canine cholera. J. Clin. Invest. 47:1210-1220. 4. Cuatrecasas, P. 1973. Affinity chromatography and structural analysis of vibrio cholerae enterotoxin-ganglioside agarose and biological effects of gangliosidecontaining soluble polymers. Biochemistry 12:42534263. 5. Deporter, D. A. 1977. Cyclic adenosine 3',5'-monophosphate and leucocyte chemotaxis in vivo. Br. J. Pharmacol. 60:205-207.

6. Deporter, D. A., F. Capasso, and D. A. Willoughby. 1976. Effects of modification of intracellular cyclic AMP levels on the immediate hypersensitivity reaction in vivo. J. Pathol. 119:147-158. 7. Estensen, R. D., H. R. Hill, P. G. Quie, N. Hogan, and N. D. Goldberg. 1973. Cyclic GMP and cell movement. Nature (London) 245:458-460. 8. Field, M. 1971. Intestinal secretion: effect of cyclic AMP and its role in cholera. New Engl. J. Med. 284:11371144. 9. Finkelstein, R. A., M. Boesman, S. H. Neoh, M. K. Larue, and R. Delaney. 1974. Dissociation and recombination of the subunits of the cholera enterotoxin (choleragen). J. Immunol. 113:145-150. 10. Gill, D. M. 1975. The arrangement of subunits in cholera toxin. Biochemistry 15:1242-1248. 11. Gill, D. M. 1978. Seven toxic peptides that cross cell membranes, p. 291-332. In J. Jeljaszewicz and T. Wadstrom (ed.), Bacterial toxins and cell membranes. Academic Press Inc., London. 12. Gill, D. M., and C. A. King. 1975. The mechanism of action of cholera toxin in pigeon erythrocyte lysates. J. Biol. Chem. 250:6434-6333. 13. Giroud, J. P., and M. Roch-Arveiller. 1977. Action du dibutyryl AMPc et du 8-bromo GMPc in vivo sur le chimiotactisme des polynucleaires de Rat. C. R. Acad. Sci. Paris 285:447-450. 14. Giroud, J. P., M. Roch-Arveiller, and 0. Muntaner. 1978. Prelevement repete des polynucleaires dans la cavity pleurale de Rat. Application a l'etude du chimiotactisme. Nouv. Rev. Fr. Hematol. 20:535-543. 15. Hewlett, E. L., R. L. Guerrant, D. J. Evans, Jr., and W. B. Greenough. 1974. Toxins of vibrio cholerae and Escherichia coli stimulate adenyl cyclase in rat fat cells. Nature (London) 249:371-373. 16. Johnson, G., W. D. Morgan, and I. Pastan. 1972. Regulation of cell motility by cyclic AMP. Nature (London) New. Biol. 235:54-56. 17. Rivkin, I., and E. L. Becker. 1976. Effect of exogenous cyclic AMP and other adenine nucleotides on neutrophil chemotaxis and motility. Int. Arch. Allergy Appl. Immunol. 50:95-102. 18. Rivkin, O., J. Rosenblatt, and E. L. Becker. 1975. The role of cyclic AMP in the chemotactic responsiveness and spontaneous motility of rabbit peritoneal neutrophils. J. Imrnunol. 115:1126-1134. 19. Schafer, D. E., W. D. Lust, B. Sircar, and N. D. Goldberg. 1970. Elevated concentration of adenosine 3':5' cyclic monophosphate in intestinal mucosa after treatment with cholera toxin. Proc. Natl. Acad. Sci. U. S. A. 67:851-856. 20. Van Heyningen, W. E. 1971. Gangliosides as membrane receptors for tetanus toxin, cholera toxin and serotonin. Nature (London) 249:415-417. 21. Willoughby, D. A., J. Dunn, S. Yamamoto, F. Capasso, D. A. Deporter, and J. P. Giroud. 1975. Calcium pyrophosphate induced pleurisy in rats: a new model of acute inflammation. Agents Actions 5:35-38. 22. Zieve, P. D., N. F. Pierce, and W. B. Greenough. 1970. Stimulation of glycogenolysis by purified cholera enterotoxin in disrupted cells. Clin. Res. 18:690.

Inhibition by cholera toxin of rat polymorphonuclear leukocyte chemotaxis demonstrated in vitro and in vivo.

INFECTION AND IMMUNITY, July 1979, p. 187-190 0019-9567/79/07-0187/04$02.00/0 Vol. 25, No. 1 Inhibition by Cholera Toxin of Rat Polymorphonuclear Le...
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