Latrunculin a is a potent inducer of aggregation of polymorphonuclear leukocytes

PI1 SOO24-3205(97)00435

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LATRUNCULIN A IS A POTENT INDUCER OF AGGREGATION POLYMORI’HONUCLEAR LEUKOCYTES

OF

Carlos A. Oliveiral, Silvana Chedraoui and Bernard0 Mantovani

Department

of Biochemistry,

RibeirIo Preto Medical School, University of SIo Paulo, 14049-900 Ribeirb Preto, S.P., Brazil (Received in final form May 8,

1997)

Summary

We have shown that latnmculin A, a toxin from a Red Sea sponge which has striking effects in several phenomena dependent on the cytoskeleton, is a potent inducer of aggregation of rabbit polymorphonuclear leukocytes in a dose- and time-dependent manner. From 12 nM to 60 nM toxin the degree of aggregation (after 8 min) in nearly directly proportional to the toxin concentration; the aggregation effect is energy-dependent from the glycolytic pathway. It was also shown that 120 nM latrunculin A, after 5 min incubation, can reduce to more than half the F-a&n percent of the leucocytes.These results may contribute to the study of the relations between the actin cytoskeleton of leukocytes and the process of aggregation which is involved in important physiological functions of these cells. Key Words: latnmculin A, polymorphonuclear leukocytes, ce.lhlar aggregation, actin microfilaments

Cellular aggregation is a component of the physiological functions of polymorphonuclear leukocytes (PMN) that must play a role in intlammatory reactions as well as in ischemic events leading to irreversible tissue lesions. Various substances can act as inducers of PMN aggregation, such as the chemotactic peptide n-formyl-methionyl-leucyl-phenylalanine (FMLP), the complement component C5a, leukotriene B4 and platelet-activating factor (1, 2). The process was shown to be accompained by some changes in the cytoskeleton, albeit no clear picture exists as yet about the mechanism of aggregation and its dependence on the structure and dynamics of the cytoskeleton network (3-6). A possible approach in this direction is to use specific drugs that interfere with the intracellular microfilaments organization, trying to correlate their effects on the cytoskeleton with the cell behaviour. Cytochalasins are among the few substances available that interfere with the actin microfihunents by binding to the growing (barbed) end of F-actin filaments(7); cytochalasin

Corresponding author: Dr. Bernard0 Mantovani, Department of Biochemistry, RibeirIo Preto Medical School, University of S%o Paulo, 14049-900 RibeirIo Preto, SP, Brazil. FAX:+55 16 633 6840. ’ Present address: Department of Chemistry, Federal University of Uberlindia.

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Aggregation of PMN by Latruncuhn A

Vol. 61, No. 6, 1997

B was shown to potentiate the aggregation response to some substances, such as the chemotactic agent FMLP, concanavalin A and phorbol myristate acetate (8) but in other conditions an inhibitory effect of cytochalasin B on leukocyte aggregation was observed (9). Another drug, which can block actin polymerization and was shown to have striking effects in various cellular phenomena is latrunculin A (10-12) a toxin produced by a Red Sea sponge (13) which we have shown to be one of the most powerful inhibitors of phagocytosis by macrophages and PMN (14,lS). This study was designed to investigate the possible action of this marine toxin as an inducer of PMN aggregation; we have also shown the effect of latrunculin A on the cellular content of polymerized actin.

Materials and Methods Reagents and media Purified latrunculin A was a gift from Dr. Y. Kashman and was prepared as described in (13). The toxin was dissolved in dimethyl sulfoxide (DMSO, Merck, Darmstadt, Germany) to a concentration of 8 mg/ml and stored at 4OC. For the biological assay this solution was diluted in Hanks’ medium to the desired concentration. Phosphate-buffered saline (PBS) containing 0,9% NaCl and 0.007 M phosphate buffer, pH 7.2, was used. DNA and DNAse I was obtained from Worthington Biochemical Corp. (Freehold, NJ, U.S.A.), and 2-deoxy-D-glucose from Sigma Chemical Co (St. Louis, MO, U.S.A.). Preparation

of PMN

Rabbits (New Zealand) were bled by cardiac puncture and the blood was collected in nearly the same volume of Alsever’s medium. PMN were isolated as described in (16). The cells were suspended in Hanks’ medium containing 0.1% gelatin. About 95% cells were PMN, and viability tests made with trypan blue indicated no more than 8% of nonviable cells. Aggregation

assay

Aggregation of PMN was measured photometrically as described in (17) in a standard aggregometer (Whole-blood Ag regometer, Chrono-Log-Corp., Havertown, Pa, U.S.A.): 0.45 ml of a suspension of cells (5.10 f?/ml) in Hanks with 0.1% gelatin was added into the cuvette, containing a teflon-coated stirring bar revolving at 900 r.p.m., and incubated for 2 min to allow warming to 370C. Then, 501 of Latnmculin A solution was added and the light transmittance recorded. To the control cuvettes 50 ul of Hanks’ medium containing 0.06% (v/v) DMSO were added. In order to study the effect of inhibition of the glycolytic pathway, the cells were preincubated with 2-deoxy-D-glucose (5.5 n&I) for 30 min at 370C before the addition of the toxin. Determination

of % F-actin in PMN

G- and F-actin of PMN were determined by the DNAse inhibition assay as described in (18). Briefly, lo6 cells in 2mI Hanks with 0.1% gelatin were added to a 9 cm plastic Petri dish, and the cells allowed to attach for 10 min at room temperature. The medium was then drained and 5 ml Hanks containing 120 nM latrunculin A or 0.06% (v/v) DMSO (control) were added and the attached cells incubated for 5 or 30 min at 370C. Detailed procedures for extraction of actin and quantification of % F-actin are given in reference (15).

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Aggregation

605

of PMN by Latrunculii A

Results The kinetic experiments of Fig. 1 show that latrunculin A can induce PMN aggregation and time-dependent manner.

in a dose-

.

J

,’

20

40

60

120

140

Latrunculin A (nM)

Fig. 1. Aggregation of PMN induced by latrunculin A (La-A). Fig 1A shows the kinetics of cellular aggregation induced by various concentrations of the toxin (t represents the variaction of light transmittance in arbitrary units). In Fig 1B the aggregation after 8 min incubation (arbitrary units, as measured by the distance in mm from the base line, in the recorder chart, discounting the initial artefact of dilution) is plotted against latrunculin A concentrations. Aggregation was measured by light transmittance with various concentrations of the toxin (the rapid initial increase of transmittance at zero time is a dilution artefact and must be discounted in quantification). Starting with 12 nM toxin up to 60 nM, the degree of aggregation, after 8 min, varies nearly linearly with latrunculin A concentration. An illustration of cellular aggregation is shown in Fig. 2. The process is clearly energy-dependent since very low aggregation results when the glycolytic pathway is blocked by incubating the cells with the toxin in Hanks’ medium without glucose and in the presence of 5.5 mM 2-deoxy-D-glucose (Fig. 3). The presence of divalent cations in the medium is also important, since no aggregation results in Hanks’ medium without Ca2+ and Mg2+ (data not shown).

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Aggregation of PMN by Latrunculin A

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Fig. 2 Phase contrast photomicrographs showing the aggregation effect of latrunculin A on PMN. Cells were incubated for 5 min at 37C either in Hanks with 0.06% DMSO (A) or with 120 nM latrunculin A (B).

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Fig. 3 The effect of 2-deoxy-D-glucose (inhibition of giycolytic pathway) on the induction of PMN aggregation by latrunculin A. Aggregation was measured as in figure lB, after 8 mm incubation at 370C. Latrunculin A was added after a preincubation of cells for 30 min at 370C with 2-deoxy-D-glucose (5.5 n&l) or other conditions as indicated. Results are mean SD (three experiments).

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Aggregationof Pht~ by Latmdin A

This marine toxin is a disrupting agent of the actin network of the cells in vitro by impairing the polymerization of pure actin in a manner consistent with the formation of a I:1 molar complex between the toxin and G-actin (19). The percent F-actin content of the cells upon the action of the toxin is shown in the experiments of Table I. The cells were incubated either with latrunculin A (120 nM) or DMSO (0.06%) as the control, for 5 and 30 min at 370C and the G- and F-actin contents were determined by the DNAse inhibition assay. After 5 min incubation the percent F-actin decreases to more than half the content of control cells, and after 30 min almost all polymerized actin has desapeared. TABLE I Effect of Latrunculin A on The F-Actin Percent of Rabbit PMN.

Conditions

% F-actin* After 5 min at 37C

After 30 min at 37C

Control (0.06% DMSO)

34.9 f 4.5 (5)

33.6 f 2.2 (3)

La-A (120 nM)

14.5 f 2.4 (9)

3.7 f 1.8 (6)

p< 0.002$

p< 0.001

* Each value represents the mean f SEM; in parenthesis, the number of independent experiments. z p values vs control (unpaired t test).

Discussion

The active concentrations of latrunculin A for several phenomena, in various types of cells, is quite variable. Thus 2.6 M impairs fertilization in sea urchins; 900 nM can disrupt the microfilaments organization of mouse fibroblast, and 90 nM has similar effects on mouse neuroblastoma cells; but as low as 20 nM is able to prevent morphogenetic movements during gastrulation in sea urchin and 30 nM can arrest cleavage at first division of its fertilized eggs (10-12). We have previously shown (14,15) that the dose-effect curve for inhibition of phagocytosis, both for macrophages and PMN (with the cells attached to glass) is situated in the range 12 to 120 nM toxin, which is nearly the same for the process of induction of aggregation of PMN in suspension. It is possible that the loss of capacity for endocytosis and the ability for cellular aggregation could be related to similar alterations in the cell membrane structure or in cytoskeleton-dependent dynamics of the membrane. The biological effects of latrunculin A are possibily related to its action as an inhibitor of actin polymerization; however, there is no clear picture on the role of actin ploymerization on PMN aggregation; conflicting results might indicate that its role is dependent on the kind of stimulus. Cytochalasin B, an inhibitor of actin microfilament assembly, can potentiate the aggregation induced by some agents (8), but in other cases it inhibits the process (9). Other experiments have shown that homotypic aggregation of human neutrophils induced by fluid shear stress correlates with F-actin accumulation in the cell-cell contact regions (20). We have shown that latrunculin A, a

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Aggregation of PMN by Latruncdin A

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blocking agent of actin polymerization, is a direct inducer of energy-dependent aggregation of rabbit PMN. This effect is possibly related to the observed cellular decrease of F-actin upon the action of this toxin. There is evidence that aggregation of leukocytes could be mediated by membrane proteins, which have been described as involved in cell to cell interaction and binding (21, 22). We can suppose that integrins or other adhesive proteins that make intracellular connections with the cytoskeleton would become more freely diffusible on the plane of the cell membrane when the cortical actin network becomes more soft upon the action of latrunculin A. Thus, the chance of these adhesive proteins to encounter the complementary binding sites on another cell would be increased, and this would result in cell aggregation. As another possibility, we may consider that leukocyte adhesiveness can be increased when local areas of the cell membrane form sections with small curvature radius, so that the cells can “roll” over their zeta potential repulsion barrier and attract one another strongly (23). If the disorganization of the cortical actin cytoskeleton would cause the formation of ridges or spiky protuberances in the membrane, this mechanism could possibily explain the aggregation effect of latrunculin A. Acknowledgements The authors wish to thank Dr. Victor Diaz Galban for the valuable help with the graphical computation, Jose A. da Silva for technical assistance and Maria Thereza Rodrigues for the expert secretarial assistance. This work was supported by the Brazilian Institution, FundaGBo de Amparo a Pesquisa do Estado de SBo Paulo. References 1. G.M. OMANN, R.A. ALLEN, G.M. BOKOCH , R.G. PAINTER , A.E. TRAYNOR and L.A. SKLAR , Physiol. Rev. 67 285-322 (1987). U. BAGGE, A MATRAl and .J.A DORMANDY, 2. E. ERNST, D.E. HAMMERSCHMIDT, JAMA 257 23 18-2324 (1987). 3. J.T. O’FLAHERTY, D.L. KREUTZER, H.J. SHOWELL and P.A. WARD, J. Immunol. 119 1751-1756 (1977). Proc. Sot. Exp. 4. J.T. O’FLAHERTY, L.R. DECHATELET, C.E. MacCALL and D.A. BASS, Biol. Med. 165 225-232 (1980). J. Immunol. 149 2549-2559 (1992). 5. L. SHAO-LEE, D. DERR and J.E.K. HILDRETH, 6. S. LABROUCHE, G. FREYBURGER, F. BELLOC and M.R. BOISSEAU, Thromb. Haemostas 68 556-562 (1992). 7 T.D. POLLARD and J.A. COOPER Ann. Rev. Biochem. 55 987-1035 (1986). 8 J.C. WHITIN and H.J. COHEN, J. Immunol. 134 1206-1211 (1985). 9 W. DE SMET, H. WALTER and L. VAN HOVE L, Immunology 79 46-54, (1993). 10 I. SPECTOR N.R. SCHOCHET, Y. KASHMAN and A. GROWEISS, Science 219 493-495 (1983). D. BLASBERGER, and Y. KASHMAN, Cell Moti.1 11 I. SPECTOR, N.R. SCHOCHET, Cytoskeleton 13 127- 144, (1989). 12 G. SCHATTEN, H. SCHATTEN, I. SPECTOR, C. CLINE, N. PAWELETZ, C. SIMERLY and C. PETZELT, Exp. Cell Res. 166 191-208 (1986). 13 Y. KASHMAN, A. GROWEISS and U. SHMUEL,I, Tetrahedron Lett 2-l 3629-3632 (1980). Life Sci. 43 1825-1830 (1988). 14 C.A. OLIVEIRA and B. MANTOVANI, 15 C.A. OLIVEIRA, Y. KASHMAN and B. MANTOVANI, Chemico-Biol. Inter. 100 141-153, (1996). 16. P.M. HENSON, J. Immunol. 107 1535-1540 (1971). 17. D.E. HAMMERSCHMIDT, Blood 55 898-902 (1980).

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