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Latrunculin A is a potent inhibitor of phagocytosis by macrophages
Life Sciences, Vol. 43, pp. 1825-1830 Printed in the U.S.A.
Pergamon Press
LATRUNCULIN A IS A POTENT IlJHIBITOR OF PHAGOCYTOSIS BY MACROPHAGES Carlos A. de Oliveira Department University
and Bernard0
Mantovani'
of Biochemistry, Ribeirao Preto Medical School of Sao Paulo, 14049 Ribeirao Preto, SP, Brazil (Receivedin final form September 30, 1988) Summary
We have found that latrunculin A, a Red Sea sponge toxin, is a potent inhibitor of immunological phagocytosis by mouse peritoneal macrophages, but does not block the binding (recognition) of the immune complexes (erythrocytes sensitized with IgG antibodies) to the cells. The inhibition begins to be appreciable around 12 nM latrunculin A, and is complete with a toxin concentration of 60 nM. This inhibitory effect does not interfere with the cell viability, and can be reversed when the macrophages are incubated in fresh medium. Since latrunculin A is a disrupting agentofmicrofilament organization, these results strenghten the evidence for the active participation of microfilaments in the mechanism of phagocytosis and at the same time provide a new tool for the investigation of phagocytosis at the molecular level. There is considerable evidence that the cytoskeletal network must be involved in phagocytosis (l-4). A wealth of information in eucaryotic cells implicates microfilaments in the mechanism of cellular motility and changes of cell shape (5,6). It is reasonable to suppose that microfilaments should play an important role in the dynamics of the phagocytic process, which requires cell membrane movements and localized changes of form. Indeed, cytochalasin B, a microfilament disrupting agent, has been found to inhibit phagocytosis in macrophages and polymorphonuclear leukocytes (7-10). Recently Kashman et al. (11) have isolated and chemically characterized latrunculins,a new class of toxins from the Red Sea sponge Latrunculia magnifica, which have been shown to interfere with the organization of microfilaments (12) and inhibit the microfilament mediated process of egg fertilization and early development in sea urchin and mice (13). In this study we investigated by in vitro experiments the effects of latrunculin A in phagocytosis of IgG-sensitized red cells by mouse peritoneal macrophages, considering the macrophage capacity for binding to
1
to whom correspondence
should be addressed.
0024-3205188$3.00 + .OO Copyright (c) 1988 Pergamon Press plc
1826
Latrunculin A
Vol. 43, h:o.22, 1988
the sensitized red cells as well as the efficiency of the phagocytes in performing the interiorization (engulfment) of these immune complexes. Materials
and llethods
Random-bred Swiss mice, 20 to 25 g body wt, were used as a source of peritoneal macrophages and of immune serum. Sheep erythrocytes were obtained from a local animal center ir Alsever's medium under sterile conditions. Phosphate buffered saline (PBS) containing 0,9% NaCl and 0.007 M phosphate buffer, pH 7.2, was used. Hanks' medium was prepared as described (14). Preparation of the immune complexes: Theimmune complexes used in the ohaqocvtosis experiments were sheep red cells covered (sensitized) with mouse IgG antibodies. The procedures for obtaining the immune serum, purification of IgG and preparation of the immune complexes are described in detail in (15). Latrunculin A: Purified latrunculin A was a gift from Dr. Y. Kashman; the methods of purification and chemical characterization are described in (11,16). It was dissolved in dimethyl sulfoxide (DMSO) and stored in the refrigerator (4 to 60~). All the controls contained the same concentrations of DMSO as the corresponding experiments with latrunculin A, and the maximum concentration of DMSO used was 0.06% (for 120 nM latrunculin A). (lo), that up We have found, in accord with other observations to 0.1% DMSO had no effect in phagocytosis. Phagocytosis experiments: The phagocytosis assays were performed with monolayers of mouse peritoneal macrophages, recently harvested, attached to glass coverslips, as described previously in detail (17). Briefly, macrophages were layered on glass coverslips and allowed to attach to glass for 10 min at room temperature; then, after washing with Hanks, the cells were incubated in plastic chambers in this medium containing the desired concentration of latrunculin A, for 30 min at 370~ in an air atmosphere saturated with water vapor. The macrophage monolay rs were then incubated with the red cell suspension (8 x 10 & red cells/r;l) in Hanks containing the same concentration of latrunculin A, for 30 min at 370C. The attached and ingested erythrocytes were differentiated by use of a hypotonic shock treatment (five times diluted PBS, 45 s) which lysed only the attached red cells. After fixation of the cells with glutaraldehyde and staining with Giemsa, the experiments were quantified by microscopic observation; at least 200 macrophages were counted in each determination. For each experimental condition, in general four independent assays (macrophages from different animals) were done. The cell viability was determined in the end of each experiment by the trypan blue exclusion method (18), in separate macrophage monolayers subjected to the same conditions of incubation. The experiment to test for the reversibility of the inhibitory effect of latrunculin A was performed as follows: after incubation o macrophages with 120 nM latrunculin A in llanks for 6 30 min at 37 C, the cells were washed with medium and incubated again with 5 ml of fresh Hanks' medium for 10 min at 37OC; after washing with Hanks this incubation was repeated once in another
LatrunculinA
vol. 43, No. 22, 1988
1827
dish. Then the phagocytosis assay was performed, as described above. Controls followed the same sequence of incubations, without latrunculin A. Results
and Discussion
The experiments of figure 1 show the effects of increasing concentrations of latrunculin A on phagocytosis of sheep erythrocytes sensitized with IgG antibodies. The macrophage monolayers were preincubated with the toxin for 30 min at 37OC and the phagocytosis experiment was performed in the presence of the toxin, incubating the r,lacrophageswith the immune complexes for 30 min at 37OC. The phagocytosis (engulfment) and the total uptake (which includes both the attached and the ingested red cells) were quantified. The latter is an index of the capacity for interaction of the sensitized red cells with the macrophages. Phagocytosis was measured by a) the percent of macrophages which were able to perfom phayocytosis (Fig. 1A) (this is an assay for the distribution of the phagocytic capacity in the cell popula(Fig. 1B) tion), and b) the ratio ingested red cell/macrophage which measures the global Fhagocytic efficiency of the cell population. The results show that latrunculin A is a potent inhibitor of phagocytosis, without interfering with cell viability. More specifically, it can block the engulfment phase, but does not impede the recognition of the immune complex (interaction) mediated by Fc receptors (there is only a small decrease in the total uptake). The inhibition of phagocytosis begins to be appreciable around 12 nM latrunculin A and is complete at a concentration of 60 nM. We have found that this inhibition by latrunculin A is reversible as shown in the experiments summarized in Table I. After preincubation of nacrophages with a high dose of the toxin (120 nM) the phagocytic capacity was restored when the cells were incubated again with fresh Hanks' medium (two successive incubations of 10 min each at 37OC); the recovery however was not complete. The fact that the binding of the immune complexes mediated by Fc receptors is not blocked by this toxin indicates that this phase must not require the participation of the intracellular microfilaments (the supposed site of action of latrunculin A, as suggested by its effects on some properties of other cells (12). A possibly similar inhibitor of phagocytosis is the drug cytochalasin B, which is also active on microfilaments.This drug, however, requires concentrations higher than 1 ug/ml (2 PM) to inhibit phagocytosis of IgG-sensitized red cells by mouse peritoneal macrophages (10). Thus, latrunculin A which at 12 nM starts the inhibitory effect is about 160 times more potent an inhibitor than cytochalasin B. The active concentrations of latrunculin A on different phenomena so far studied in various types of cells is quite variable (12,13).Thus, the impairment of sea urchin fertilization requires 2.6 ~11; 900 nM can disrupt the microfilament organization of mouse fibroblasts; 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
1828
Vol. 43, so. 22, 1988
LatrunculinA
0
12
24
36
48
LATRUNCULIN
60" 120
A in M 1
E'IG. 1 The effect of latrunculin A on phagocytosis by mouse peritoneal macrophages. Macrophages were incubated with increasing concentrations of latrunculin A, for 30 min at 370C. Thereafter the immune complexes (sheep red cells sensitized with mouse IgG antibodies) were incubated with the macrophages for 30 min at 37OC, in the presence of the corresponding concentrations of the toxin. Quantification of the experiment was done by the measurement of a) the percent of the number of macrophages counted which had ingested at least one red cell (A, percent phagocytosis); b) the number of ingested erythrocytes divided by the total number, of macrophages counted (B, red cell/macrophage); c) the considering all same ratio (red cell/macrophage) (attached plus ingested) red cells (B, total uptake). Viability (A) represents the percent of macrophages that excludes the trypan blue-Results are means f SE (four animals).
LatrunculinA
Vol. 43, No. 22, 1988
1829
33 nM can block the cleavage at first division of its fertilized eggs. One can see that the concentrations of latrunculina A we have found to inhibit phagocytosis (12 nM to 60 nM) places this phenomenon among the most suscetible ones to this marine toxin. TABLE Reversibility
I
of the Inhibitory Effect of Latrunculin Phagocytosis by Macrophages
Phagocytosis
Total uptake
Condition percent Control
78 * 2
LatrunculinAa1.2 ? 0.1 (120 nM) LatrunculinAb 48 + 3 followed by fresh medium
C rc/mac
rc/macc 2.2
+
0.22
A on
Viability percent
9.9
+ 0.23
96 ? 0.4
0.012 2 0.001
7.9
? 0.14
95 : 0.6
0.74
8.7
+ 0.27
96 ? 0.7
2 0.06
Note. Quantification was performed as in figure 1. All data are means + SE (four animals). a After preincubation of raacrophages with 120 nM latrunculin A (30 min at 37OC) the phagocytosis assay was readily done as in figure 1. b After preincubation of macrophages with 120 nM latrunculin A (30min at 37OC) the cells were washed and incubated with 5 ml of fresh Hanks' medium (without the toxin) for 10 min at 370C; this incubation was repeated onceinanother dish, and the phagocytosis assay was performed (for 30 min at 37OC). Controls followed the same sequence of incubations (without latrunculin A). C red cell/macrophage. The participation of actin microfilaments in the mechanism of phagocytosis has been suggested by a number of observations
in amoebae, macrophages and polymorphonuclear leukocytes (l-3). Recently it has been shown with neutrophil leukocytes that an early event, after recognition of the immune complex, is a rapid (less than 10 s) and transient polymerization of actin; the microfilaments associated with the phagosomes however are dispersed shortly after internalization (4). It is possible that latrunculin A blocks phagocytosis in this post-recognition since some observations show that this actin polymerization, marine toxin is able to form a complex with G-actin, inhibiting its polymerization to F-actin (19). However, the concentration of latrunculin A required was much higher than is needed to inhibit phagocytosis, i.e. 2-12 uM (with a dissociation constant of 0.2 uM) compared to 12-60 nM. Of course, it is possible that the cells concentrate the toxin. But how, precisely, the contractile network of cytoskeleton functions in phagocytosis is yet a challenge for investigation. Although
one cannot exclude
the possibility
that latrunculin microfilament with the known biochemical
A might have an effect on macrophages otherthan.on organization,
our results
together
1830
Latrunculin
A
Vol.
43,
No.
22,
1988
properties of this toxin, strenghten the evidence for an active role of microfilamentsinphagocytosis, and at the same time provide a new tool for the investigation of phagocytosis at the molecular level. Aknowledgments The authors thank Dr. Y. Kashman for the generous gift of latrunculin A, Jo& Antonio da Silva for technical assistance and Maria Theresa Rodrigues for secretarial assistance. This research had the support of the following brazilian institutions: FAPESP, FINEP and CNPq. References 1. E.D. KORN, B. BOWERS, S. BATZRI, S.R. SIMMONS and E.J. VICTORIA, J. Supranol. Strut. 2 517-528 (1974). 2. R.D. BERLIN and J.M. OLIVER, JT Cell Biol. 77 789-804 (1978). 3. 0.1. STENDAHL, J.H. HARTWIG, E.A. BROTSCHI and T.P. STOSSEL, J. Cell Biol. 84 215-224 (1980). RICKAEW and R.C. RICHARDS, Eur. J. Cell 4. P. SHETERLINE,?.E. Biol. 34 80-87 (1984). 5. M. CL.AEE and J.A. SPUDICH, Ann. Rev. Biochem. -46 797-822 (1977). 6. H.M. WARRICK and J.A. SPUDICH, Ann. Rev. Cell Biol. 3 379-421 (1987). 7. A.T. DAVIS, R. ESTENSEN and P.G. QUIE, Proc. Sot. Exptl. Biol. Med. 137 161-164 (1971). 8. A.C. ALLISON, P. DAVIES and S.DE PETRIS, Nature New Biol. 232 153-154 (1971). 9. S.H. ZIGMOND and J.G. HIRSCH, Exp. Cell Res. -73 383-393 (1972). 10. G.G. KLAUS, Exp. Cell Res. 79 73-78 (1973). 11. Y. KASHMAN, A. GROWEISS and3. SHMUELI, Tetrahedron Lett. 21 3629-3632 (1980). 12. I. SPECTOR, N.R. SCHOCHET, Y. KASHMAN and A. GROWEISS, Science 214 493-495 (1983). H. SCHATTEN, I. SPECTOR, C. CLINE, N. PAWELETZ, 13. G. SCHATS, C. SIMERLY and C. PETZELT, Exp. Cell Res. 166 191-208 (1986). 14. J. PAUL, Cell and Tissue Culture, 4th ed., p. 86 Livingstone, Edinburg/London (1970). 15. B. MANTOVANI, 1.1.RABINOVITCH and V. NUSSENZWEIG, J. Exp. Med. 135 780-792 (19721. 16. CNEEMAN, L: ~IsiimsoEj and 1. KASHMAN, Marine Bioloqy -30 293-297 (1975). Cell Res. 173 282-286 (1987). 17. B. MANTOVANI,.Exp. 18. E.A. BOYSE, L.J. OLD and I. CHOUROULINKOV, in Methods in Medical Research (H-N. Eiscn, Ed.) Vol. 10,pp 39-47, Year Book Medical Publishers, USA (1964). 19. M. COUE, S.L. BRENNER, I. SPECTOR and E.D. KORN, FEBS Lett. 213 316-318 (1987).
Pergamon Press
LATRUNCULIN A IS A POTENT IlJHIBITOR OF PHAGOCYTOSIS BY MACROPHAGES Carlos A. de Oliveira Department University
and Bernard0
Mantovani'
of Biochemistry, Ribeirao Preto Medical School of Sao Paulo, 14049 Ribeirao Preto, SP, Brazil (Receivedin final form September 30, 1988) Summary
We have found that latrunculin A, a Red Sea sponge toxin, is a potent inhibitor of immunological phagocytosis by mouse peritoneal macrophages, but does not block the binding (recognition) of the immune complexes (erythrocytes sensitized with IgG antibodies) to the cells. The inhibition begins to be appreciable around 12 nM latrunculin A, and is complete with a toxin concentration of 60 nM. This inhibitory effect does not interfere with the cell viability, and can be reversed when the macrophages are incubated in fresh medium. Since latrunculin A is a disrupting agentofmicrofilament organization, these results strenghten the evidence for the active participation of microfilaments in the mechanism of phagocytosis and at the same time provide a new tool for the investigation of phagocytosis at the molecular level. There is considerable evidence that the cytoskeletal network must be involved in phagocytosis (l-4). A wealth of information in eucaryotic cells implicates microfilaments in the mechanism of cellular motility and changes of cell shape (5,6). It is reasonable to suppose that microfilaments should play an important role in the dynamics of the phagocytic process, which requires cell membrane movements and localized changes of form. Indeed, cytochalasin B, a microfilament disrupting agent, has been found to inhibit phagocytosis in macrophages and polymorphonuclear leukocytes (7-10). Recently Kashman et al. (11) have isolated and chemically characterized latrunculins,a new class of toxins from the Red Sea sponge Latrunculia magnifica, which have been shown to interfere with the organization of microfilaments (12) and inhibit the microfilament mediated process of egg fertilization and early development in sea urchin and mice (13). In this study we investigated by in vitro experiments the effects of latrunculin A in phagocytosis of IgG-sensitized red cells by mouse peritoneal macrophages, considering the macrophage capacity for binding to
1
to whom correspondence
should be addressed.
0024-3205188$3.00 + .OO Copyright (c) 1988 Pergamon Press plc
1826
Latrunculin A
Vol. 43, h:o.22, 1988
the sensitized red cells as well as the efficiency of the phagocytes in performing the interiorization (engulfment) of these immune complexes. Materials
and llethods
Random-bred Swiss mice, 20 to 25 g body wt, were used as a source of peritoneal macrophages and of immune serum. Sheep erythrocytes were obtained from a local animal center ir Alsever's medium under sterile conditions. Phosphate buffered saline (PBS) containing 0,9% NaCl and 0.007 M phosphate buffer, pH 7.2, was used. Hanks' medium was prepared as described (14). Preparation of the immune complexes: Theimmune complexes used in the ohaqocvtosis experiments were sheep red cells covered (sensitized) with mouse IgG antibodies. The procedures for obtaining the immune serum, purification of IgG and preparation of the immune complexes are described in detail in (15). Latrunculin A: Purified latrunculin A was a gift from Dr. Y. Kashman; the methods of purification and chemical characterization are described in (11,16). It was dissolved in dimethyl sulfoxide (DMSO) and stored in the refrigerator (4 to 60~). All the controls contained the same concentrations of DMSO as the corresponding experiments with latrunculin A, and the maximum concentration of DMSO used was 0.06% (for 120 nM latrunculin A). (lo), that up We have found, in accord with other observations to 0.1% DMSO had no effect in phagocytosis. Phagocytosis experiments: The phagocytosis assays were performed with monolayers of mouse peritoneal macrophages, recently harvested, attached to glass coverslips, as described previously in detail (17). Briefly, macrophages were layered on glass coverslips and allowed to attach to glass for 10 min at room temperature; then, after washing with Hanks, the cells were incubated in plastic chambers in this medium containing the desired concentration of latrunculin A, for 30 min at 370~ in an air atmosphere saturated with water vapor. The macrophage monolay rs were then incubated with the red cell suspension (8 x 10 & red cells/r;l) in Hanks containing the same concentration of latrunculin A, for 30 min at 370C. The attached and ingested erythrocytes were differentiated by use of a hypotonic shock treatment (five times diluted PBS, 45 s) which lysed only the attached red cells. After fixation of the cells with glutaraldehyde and staining with Giemsa, the experiments were quantified by microscopic observation; at least 200 macrophages were counted in each determination. For each experimental condition, in general four independent assays (macrophages from different animals) were done. The cell viability was determined in the end of each experiment by the trypan blue exclusion method (18), in separate macrophage monolayers subjected to the same conditions of incubation. The experiment to test for the reversibility of the inhibitory effect of latrunculin A was performed as follows: after incubation o macrophages with 120 nM latrunculin A in llanks for 6 30 min at 37 C, the cells were washed with medium and incubated again with 5 ml of fresh Hanks' medium for 10 min at 37OC; after washing with Hanks this incubation was repeated once in another
LatrunculinA
vol. 43, No. 22, 1988
1827
dish. Then the phagocytosis assay was performed, as described above. Controls followed the same sequence of incubations, without latrunculin A. Results
and Discussion
The experiments of figure 1 show the effects of increasing concentrations of latrunculin A on phagocytosis of sheep erythrocytes sensitized with IgG antibodies. The macrophage monolayers were preincubated with the toxin for 30 min at 37OC and the phagocytosis experiment was performed in the presence of the toxin, incubating the r,lacrophageswith the immune complexes for 30 min at 37OC. The phagocytosis (engulfment) and the total uptake (which includes both the attached and the ingested red cells) were quantified. The latter is an index of the capacity for interaction of the sensitized red cells with the macrophages. Phagocytosis was measured by a) the percent of macrophages which were able to perfom phayocytosis (Fig. 1A) (this is an assay for the distribution of the phagocytic capacity in the cell popula(Fig. 1B) tion), and b) the ratio ingested red cell/macrophage which measures the global Fhagocytic efficiency of the cell population. The results show that latrunculin A is a potent inhibitor of phagocytosis, without interfering with cell viability. More specifically, it can block the engulfment phase, but does not impede the recognition of the immune complex (interaction) mediated by Fc receptors (there is only a small decrease in the total uptake). The inhibition of phagocytosis begins to be appreciable around 12 nM latrunculin A and is complete at a concentration of 60 nM. We have found that this inhibition by latrunculin A is reversible as shown in the experiments summarized in Table I. After preincubation of nacrophages with a high dose of the toxin (120 nM) the phagocytic capacity was restored when the cells were incubated again with fresh Hanks' medium (two successive incubations of 10 min each at 37OC); the recovery however was not complete. The fact that the binding of the immune complexes mediated by Fc receptors is not blocked by this toxin indicates that this phase must not require the participation of the intracellular microfilaments (the supposed site of action of latrunculin A, as suggested by its effects on some properties of other cells (12). A possibly similar inhibitor of phagocytosis is the drug cytochalasin B, which is also active on microfilaments.This drug, however, requires concentrations higher than 1 ug/ml (2 PM) to inhibit phagocytosis of IgG-sensitized red cells by mouse peritoneal macrophages (10). Thus, latrunculin A which at 12 nM starts the inhibitory effect is about 160 times more potent an inhibitor than cytochalasin B. The active concentrations of latrunculin A on different phenomena so far studied in various types of cells is quite variable (12,13).Thus, the impairment of sea urchin fertilization requires 2.6 ~11; 900 nM can disrupt the microfilament organization of mouse fibroblasts; 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
1828
Vol. 43, so. 22, 1988
LatrunculinA
0
12
24
36
48
LATRUNCULIN
60" 120
A in M 1
E'IG. 1 The effect of latrunculin A on phagocytosis by mouse peritoneal macrophages. Macrophages were incubated with increasing concentrations of latrunculin A, for 30 min at 370C. Thereafter the immune complexes (sheep red cells sensitized with mouse IgG antibodies) were incubated with the macrophages for 30 min at 37OC, in the presence of the corresponding concentrations of the toxin. Quantification of the experiment was done by the measurement of a) the percent of the number of macrophages counted which had ingested at least one red cell (A, percent phagocytosis); b) the number of ingested erythrocytes divided by the total number, of macrophages counted (B, red cell/macrophage); c) the considering all same ratio (red cell/macrophage) (attached plus ingested) red cells (B, total uptake). Viability (A) represents the percent of macrophages that excludes the trypan blue-Results are means f SE (four animals).
LatrunculinA
Vol. 43, No. 22, 1988
1829
33 nM can block the cleavage at first division of its fertilized eggs. One can see that the concentrations of latrunculina A we have found to inhibit phagocytosis (12 nM to 60 nM) places this phenomenon among the most suscetible ones to this marine toxin. TABLE Reversibility
I
of the Inhibitory Effect of Latrunculin Phagocytosis by Macrophages
Phagocytosis
Total uptake
Condition percent Control
78 * 2
LatrunculinAa1.2 ? 0.1 (120 nM) LatrunculinAb 48 + 3 followed by fresh medium
C rc/mac
rc/macc 2.2
+
0.22
A on
Viability percent
9.9
+ 0.23
96 ? 0.4
0.012 2 0.001
7.9
? 0.14
95 : 0.6
0.74
8.7
+ 0.27
96 ? 0.7
2 0.06
Note. Quantification was performed as in figure 1. All data are means + SE (four animals). a After preincubation of raacrophages with 120 nM latrunculin A (30 min at 37OC) the phagocytosis assay was readily done as in figure 1. b After preincubation of macrophages with 120 nM latrunculin A (30min at 37OC) the cells were washed and incubated with 5 ml of fresh Hanks' medium (without the toxin) for 10 min at 370C; this incubation was repeated onceinanother dish, and the phagocytosis assay was performed (for 30 min at 37OC). Controls followed the same sequence of incubations (without latrunculin A). C red cell/macrophage. The participation of actin microfilaments in the mechanism of phagocytosis has been suggested by a number of observations
in amoebae, macrophages and polymorphonuclear leukocytes (l-3). Recently it has been shown with neutrophil leukocytes that an early event, after recognition of the immune complex, is a rapid (less than 10 s) and transient polymerization of actin; the microfilaments associated with the phagosomes however are dispersed shortly after internalization (4). It is possible that latrunculin A blocks phagocytosis in this post-recognition since some observations show that this actin polymerization, marine toxin is able to form a complex with G-actin, inhibiting its polymerization to F-actin (19). However, the concentration of latrunculin A required was much higher than is needed to inhibit phagocytosis, i.e. 2-12 uM (with a dissociation constant of 0.2 uM) compared to 12-60 nM. Of course, it is possible that the cells concentrate the toxin. But how, precisely, the contractile network of cytoskeleton functions in phagocytosis is yet a challenge for investigation. Although
one cannot exclude
the possibility
that latrunculin microfilament with the known biochemical
A might have an effect on macrophages otherthan.on organization,
our results
together
1830
Latrunculin
A
Vol.
43,
No.
22,
1988
properties of this toxin, strenghten the evidence for an active role of microfilamentsinphagocytosis, and at the same time provide a new tool for the investigation of phagocytosis at the molecular level. Aknowledgments The authors thank Dr. Y. Kashman for the generous gift of latrunculin A, Jo& Antonio da Silva for technical assistance and Maria Theresa Rodrigues for secretarial assistance. This research had the support of the following brazilian institutions: FAPESP, FINEP and CNPq. References 1. E.D. KORN, B. BOWERS, S. BATZRI, S.R. SIMMONS and E.J. VICTORIA, J. Supranol. Strut. 2 517-528 (1974). 2. R.D. BERLIN and J.M. OLIVER, JT Cell Biol. 77 789-804 (1978). 3. 0.1. STENDAHL, J.H. HARTWIG, E.A. BROTSCHI and T.P. STOSSEL, J. Cell Biol. 84 215-224 (1980). RICKAEW and R.C. RICHARDS, Eur. J. Cell 4. P. SHETERLINE,?.E. Biol. 34 80-87 (1984). 5. M. CL.AEE and J.A. SPUDICH, Ann. Rev. Biochem. -46 797-822 (1977). 6. H.M. WARRICK and J.A. SPUDICH, Ann. Rev. Cell Biol. 3 379-421 (1987). 7. A.T. DAVIS, R. ESTENSEN and P.G. QUIE, Proc. Sot. Exptl. Biol. Med. 137 161-164 (1971). 8. A.C. ALLISON, P. DAVIES and S.DE PETRIS, Nature New Biol. 232 153-154 (1971). 9. S.H. ZIGMOND and J.G. HIRSCH, Exp. Cell Res. -73 383-393 (1972). 10. G.G. KLAUS, Exp. Cell Res. 79 73-78 (1973). 11. Y. KASHMAN, A. GROWEISS and3. SHMUELI, Tetrahedron Lett. 21 3629-3632 (1980). 12. I. SPECTOR, N.R. SCHOCHET, Y. KASHMAN and A. GROWEISS, Science 214 493-495 (1983). H. SCHATTEN, I. SPECTOR, C. CLINE, N. PAWELETZ, 13. G. SCHATS, C. SIMERLY and C. PETZELT, Exp. Cell Res. 166 191-208 (1986). 14. J. PAUL, Cell and Tissue Culture, 4th ed., p. 86 Livingstone, Edinburg/London (1970). 15. B. MANTOVANI, 1.1.RABINOVITCH and V. NUSSENZWEIG, J. Exp. Med. 135 780-792 (19721. 16. CNEEMAN, L: ~IsiimsoEj and 1. KASHMAN, Marine Bioloqy -30 293-297 (1975). Cell Res. 173 282-286 (1987). 17. B. MANTOVANI,.Exp. 18. E.A. BOYSE, L.J. OLD and I. CHOUROULINKOV, in Methods in Medical Research (H-N. Eiscn, Ed.) Vol. 10,pp 39-47, Year Book Medical Publishers, USA (1964). 19. M. COUE, S.L. BRENNER, I. SPECTOR and E.D. KORN, FEBS Lett. 213 316-318 (1987).