486
Preliminary
notes
mitotic index at 48 h and no appreciable change in the fraction of micronucleate cells during incubation at 39°C (fig. 3b). These results indicate that ts 39 cells are arrested transiently at the mitotic phase and these cells are subsequently converted to multimicronucleate cells. In this connection, it is noteworthy that colcemid induces accumulation of mitotic cells which in turn undergo micronucleation [2]. Fig. 2d shows the micronucleation which was induced with colcemid in wild-type cells. There is a close resemblance in the time of appearance as well as in the morphology between temperature-induced micronuclei in ts 39 cells and colcemid-induced micronuclei in wildtype cells. These findings suggest that ts 39 cells may be deficient in microtubule assembly at the non-permissive temperature. The present mutant appears to be different from ts 2 cells which are defective in karyo- and cytokinesis [4], since ts 2 cells show unequal chromosome separation among daughter nuclei but never undergo micronucleation.
Printed in Sweden Copyright @ 1978 by Academic Press, Inc. All tights of reproduction in any form reserved 0014-4827/78/l 142-0486$02.00/O
Manipulations of the phagosome-lysosome fusion response in cultured macrophages. Enhancement of fusion by chloroquine and other amines P. D’ARCY HART and M. R. YOUNG, Nationul Institute for Medical NW7 IAA, UK
Research,
Mill
Hill,
London
The fusion of secondary lysosomes of cultured normal mouse peritoneal macrophages with phagosomes containing ingested Saccharomyces cerevisiae is inhibited by polyanions previously incorporated in the medium. In contrast, this fusion process can be accelerated by chloroquine and some other secondary and tertiary amines; and these compounds can reverse the inhibition induced by a polyanion. The non-fusion pattern usually associated with intracellular Mycobacterium tuberculosis can also be reversed by chloroquine. Our observations offer a possible new approach to the study of subcellular membrane fusion, and of factors influencing the course of experimental intracellular infections. Summary.
It is still uncertain what role the lysosomal system plays in determining whether microorganisms ingested by macrophages survive or die. Many species of microorganism that are phagocytosed by macrophages are The authors wish to thank Miss Naoko Hieda for her rapidly exposed to the contents of the lysotechnical assistance, Dr T. Shiomi for discussion, and somes as a result of phagosome-lysosome Mr R. L. Simmons for improving English. This work was supported in part by grants-in-aid from the Min- fusion (P-LF). Some pathogens, notably istry of Education, Science and Culture, Japan. virulent Mycobacterium tuberculosis [I] and living Toxoplasma gondii [2] are excepReferences tions; in cultured normal macrophages P1. Schmid, W, Mutat res 3 1 (1975) 9. 2. Ege, T & Ringer& N R, Exp cell res 87 (1974) LF is inhibited after phagocytosis. 378. It seemed possible that evasion of con3. Johnson, R T, Mullinger, A M & Skaer, R J, Proc roy sot B 189(1975) 591. tact with lysosomal contents might assist in4. Shiomi, T & Sato, K, Exp cell res 100(1976) 297. tracellular survival of these microorganS. Deitch, A D, Introd quant cytochem 1 (1966) 327. isms. Attachment of antibody to them beReceived January 18, 1978 fore their ingestion reversed the non-fusion Revised version received March 8, 1978 Accepted March 13, 1978 patterns, and led to killing and digestion of Toxoplasma [3, 41, but the multiplication of tubercle bacilli was uninhibited [5]. We sought other ways of controlling P-LF, so as to learn more about its mechanism and its functions in intracellular infections. Sev-
Preliminary
notes
487
Table 1. Proportion of yeast-containing phagosomes showing fusion with AO-labelled lysosomes in macrophages exposed to chloroquine diphosphate (10 pg ml-’ for 30 min) following ingestion of irradiated yeasts
Untreated (controls) Chloroquine-treated
% with intraphagosomal orange “rims””
% with intraphagosomal coloured yeastsb
Total % phagosomes fused
60 20
10 75
70 95
n Confluent rims of A0 fluorescence within the phagosomes and surrounding the enclosed yeasts (i.e. early stage of fusion). * Penetration of the yeasts by the A0 (i.e. later stage of fusion).
era1polyanionic substances were found that were taken up from the culture medium by endocytosis and accumulated in secondary lysosomes, and which inhibited the fusion of the latter (though probably not of primary lysosomes) with phagosomes containing yeast cells (which normally promote PLF [6, 71). We now report, in contrast, enhancement of the P-LF response by the antimalarial drug chloroquine, and preliminary results with some other secondary and tertiary amines. Added to cell culture medium, chloroquine enters the cell, probably by diffusion, and is rapidly concentrated in the lysosomes [g-l 11.We have assessedthe extent of P-LF by dark-field vital fluorescence microscopy and by electron microscopy r&71.
Materials
and Methods
Fluorescence microscopy (FM): celeration of P-LF (yeast target).
Assessment
of ac-
Normal mouse peritoneal macrophages, established as monolayers in Chang medium on coverslips in Leighton tubes [ 1, 5, 121for 1-2 weeks, were given a change of medium, and used l-5 days later. The monolayers were washed with balanced salt solution (BSS) and exposed at 37°C to acridine orange (AO, 5 WEml-‘) in BSS for 10 min (to prelabel secondary lysoiomes) [6, 71. The cells were then washed and incubated at 37°C with a suspension in BSS of commercial baker’s yeast, Saccharomyces cerevisiae (5~ lo8 yeast cells ml-l). used either fresh or after exposure to 150 krad gamma radiation (giving a survival c 1%). After 15 or 30 min the macrophages were washed and exposed at 37°C in BSS to chloroquine diphosphate (ICI Pharmaceuticals), usually 10 pg ml-’ (20 PM) for 30 or 45 min;
this dose was well tolerated by the cells (l-2 weeks). Control tubes without the drug were incubated in parallel. Then, after a final wash the coverslips were examined under blue-violet light (within 60 min after completion of the yeast ingestion). The extent of PLF was judged by observing passage of the fluorescent lysosomal marker (AO) into yeast-containing phagosomes [6, 71. The results were similar if the macrophages were exposed to the drug before the A0 labelling and subsequent ingestion of yeasts, but were less consistent. FM: Assessment of reversal qf inhibition of P-LF (yeast target). The procedure wasas above except that
poly-n-glutamic acid (PGA) (sodium salt, 100pg ml-‘) had been incorporated in fresh culture medium for 5 days; the monolayers were then washed, exposed in turn to AO, live or irradiated yeasts, and chloroquine. Change of the sequence from PGA-AG-veasts-chloroquine-to PGA-chloroquine-AG-yeasts did not affect results. Electron of inhibition
microscopy (EM): Assessment of reversal of P-LF (yeast target). The macrophage
monolayers were exposed to ferritin (10 mg ml-‘) for 3 h (to prelabel secondary lysosomes) [ 1, fi. The next day, PGA (100 ~g ml-‘) was added in fresh medium and the tubes were incubated for 5 days at 37 “C. The cultures were washed, irradiated veasts added. and the tubes reincubated for 30 min. After further ‘washing of cells, chloroquine diphosphate (10 +g ml-‘) was added to half the tubes. The macrophages were fixed after 60 min of further incubation [ 1, 131. EM: Reversal of inhibition (Mycobacterium tuberculosis target). Ferritin-prelabelled macrophages were
exposed at 37°C for 90 min to a suspension of strain H37Rv [ 11. After washing, chloroquine diphosphate (20 pg ml-‘) was added to half the tubes, and all were reincubated for 45 min. The cultures were again washed, incubated in fresh BSS for a further 60 min. and then fixed.
Results and Discussion
Acceleration of the phagosome-lysosome fusion process, judged by FM with yeasts as target, occurred in chloroquine-treated Erp Cd/ Rrs i 14 f 1978)
488
Preliminary
notes
Table 2. Proportion
of yeast-containing phagosomes showing fusion with AO-labelled lysosomes in macrophages pretreatedfor 5 days with poly-o-glutamic acid (100 pg ml-‘); then permitted to ingest irradiated yeasts; andfinally exposed to chloroquine diphosphate (10 p.g ml-‘for 30 min) PGA treatment
Chloroquine treatment
% with intraphagosomal orange “rims””
% with intraphagosomal coloured yeasts*
Total % phagosomes fused
No Yes Yes
No No Yes
55 0 50
15 0 15
70 0 65
a,* See table I.
macrophages compared with controls (table 1). Chloroquine was found also to reverse the inhibition of P-LF induced in macrophages pretreated with a polyanion, the results of a typical experiment being shown in table 2. If the PGA concentration was raised to 1.0 mg ml-’ the reversal was partial, but if the chloroquine concentration was also raised to 20 pg ml-’ for 45 min, reversal was fully restored. The reversal by chloro&ine of the PGAinduced inhibition of the P-LF response to yeasts was also shown by EM. In thin sections of cells treated with PGA alone, ferritin label was not seen in any of 200 phagosomes counted, 90% of which were of the “loose” variety [6, 71. In the macrophages treated with PGA and chloroquine, ferritin was present in 20% of 250 phagosomes counted. These differences, indicating partial reversal of inhibition, were significant (P~0.01). All ferritin-containing phagosomes were of the “tight” variety: where non-fusion had not been reversed (80%), the phagosome membranes were “tight” or “loose” in equal proportions. ‘The effect of chloroquine on the P-LF response to infection with M. tuberculosis was also examined by electron microscopy. The typical non-fusion pattern of lysosomal response to virulent M. tuberculosis [ 11was Exp Cd RPS I14 (1978)
almost completely reversed by the drug. Ferritin was seen in only about one-fifth .of phagosomes containing intact bacilli in the control macrophages, but in three-quarters of them in chloroquine-treated cells. At this dose the drug characteristically [14] induced many autophagic vacuoles, which frequently contained ferritin as well as debris and were amalgamated with the bacillus-containing phagolysosomes. (After exposure to the more usual concentration of 10 pg ml-’ (see above), autophagic vacuoles were inconspicuous.) S. cerevisiae, exposed in vitro at 37°C for 30 min to a high concentration of chloroquine diphosphate (1 .O mg ml-‘), binds the drug (demonstrable by fluorescence) and is killed. If yeasts so treated, and washed free of unbound drug, are ingested by normal macrophages there is enhancement of PLF; and if ingested by PGA-pretreated macrophages the usual inhibition of P-LF observed after ingestion of normal yeasts by PGA-treated cells is absent-both situations produced with normal yeasts only after treatment of these macrophages with free chloroquine. During the exposure of macrophages (3U5 min) to chloroquine after they have ingested normal yeasts the drug will begin to enter the yeast-containing phagosomes along with other lysosomal
Preliminary
contents. The question arises, therefore, whether both the enhancement of P-LF in normal macrophages by chloroquine and its ability to reverse the inhibition of P-LF in PGA-pretreated macrophages might be due to the presence of chloroquine bound to killed yeasts within the phagosomes and not to this drug accumulated in the lysosomes and acting at that site. However, in experiments where PGA-pretreated monolayers had ingested normal live yeasts, and were then exposed to chloroquine (10 pg ml-’ for 30 min), culture counts of the intracellular yeasts (after disruption of the macrophages by gentle ultrasonic vibration) revealed little or no loss of viability over this limited period. Furthermore, a chloroquine-resistant strain of S. cerevisiae behaved altogether like chloroquine-sensitive yeasts: PLF in normal macrophages was enhanced, and its inhibition in PGA-pretreated macrophages was reversed, only when the macrophages were exposed additionally to free chloroquine in the usual way. This strain did not bind chloroquine after exposure for 30 min in vitro to 1.0 mg ml-‘, nor was it killed by this exposure. We conclude that both the enhancement and the inhibitionreversal effects on P-LF by chloroquine are direct effects upon the cell, rather than because the drug becomes attached to the intraphagosomal yeast. The reversal by chloroquine of PGA-induced inhibition of P-LF might result from direct ionic interaction of chloroquine with the polyanion in the secondary lysosomes: however, chloroquine on its own is able to enhance fusion above normal levels. We conclude that the compounds act competitively on the cells. Some other tertiary and secondary amines have been similarly examined for their ability to accelerate the P-LF response to ingestion of yeasts in normal macro-
notes
489
phages, and to reverse its inhibition induced in polyanion (PGA)-pretreated macrophages. The following additional compounds were found to be active in both respects: quinacrine dimethane sulphonate, 3-quinuclidinyl benzilate, atropine sulphate, tetracaine hydrochloride, tri-n-butylamine and di-n-butylamine hydrochlorides (quinacrine and chloroquine being the most active on a molar basis). Triethylamine hydrochloride was weakly active, and N,Ndimethylaniline inactive. In separate experiments chloroquine, tetracaine and tributylamine have been tested for their effect on the progress of a tuberculous infection within the cultured macrophages. Each of these compounds inhibited or suppressed intracellular bacillary multiplication. The identification of some chemical effector agents able to control P-LF experimentally may offer another approach to the study of the mechanism of this membrane fusion process, as well as to the possibility of influencing the course of a pathogenic infection by this type of manipulation. We thank Drs J. A. Armstrong and P. Draper for advice and encouragement throughout this investigation; Drs G. H. Beaven, R. H. Gigg, N. J. M. Birdsall and D. C. Warhurst for technical suggestions; and Dr D. H. Williamson for the chloroquine-resistant strain of S. cerevisiae.
References 1. Armstrong, J A & Hart, P D’Arcy, J exp med 134 (1971) 713. 2. Jones, T C & Hirsch, J G, J exp med 136 (1972) 1173. 3. Jones, T C, Jreticuloendothelial sot 15(1974)439. 4. Jones, T C, Len, L & Hirsch, J G, J exp med 141 (1975) 466. 5. Armstrong, J A & Hart, P D’Arcy, J exp med 142 (1975) 1. 6. Hart, P D’Arcy & Young, M R, Nature 256 (1975) 47. 7. Goren, M B, Hart, P D’Arcy, Young, M R & Armstrong, J A, Proc natl acad sci US 73 (1976) 2510. 8. Allison, A C &Young, M R, Life sci 3 (1964) 1407. Exp Cell Res I14 (1978)
490
Preliminary notes
9. de Duve, C, de Barsy, T, Poole, B, Trouet, A, Tulkens, P & Van Hoof, F, Biochem pharmacol23 (1974) 2495. IO. Wibo, M & Poole, B, J cell bio163 (1974) 430. 11. Reijngoud, D J & Tager, J M, FEBS lett 64 (1976) 231. 12. Hart, P D’Arcy, Science 162(1968) 686.
Exp Cell Res 114 (1978)
13. Hirsch, J G & Fedorko, M E, J cell biol 38 (1968) 615. 14. Fedorko, M E, Hirsch, J G & Cohn. 2 A, J cell bio138 (1%8) 377. Received January 20, 1978 Accepted February 16, 1978
Preliminary
notes
mitotic index at 48 h and no appreciable change in the fraction of micronucleate cells during incubation at 39°C (fig. 3b). These results indicate that ts 39 cells are arrested transiently at the mitotic phase and these cells are subsequently converted to multimicronucleate cells. In this connection, it is noteworthy that colcemid induces accumulation of mitotic cells which in turn undergo micronucleation [2]. Fig. 2d shows the micronucleation which was induced with colcemid in wild-type cells. There is a close resemblance in the time of appearance as well as in the morphology between temperature-induced micronuclei in ts 39 cells and colcemid-induced micronuclei in wildtype cells. These findings suggest that ts 39 cells may be deficient in microtubule assembly at the non-permissive temperature. The present mutant appears to be different from ts 2 cells which are defective in karyo- and cytokinesis [4], since ts 2 cells show unequal chromosome separation among daughter nuclei but never undergo micronucleation.
Printed in Sweden Copyright @ 1978 by Academic Press, Inc. All tights of reproduction in any form reserved 0014-4827/78/l 142-0486$02.00/O
Manipulations of the phagosome-lysosome fusion response in cultured macrophages. Enhancement of fusion by chloroquine and other amines P. D’ARCY HART and M. R. YOUNG, Nationul Institute for Medical NW7 IAA, UK
Research,
Mill
Hill,
London
The fusion of secondary lysosomes of cultured normal mouse peritoneal macrophages with phagosomes containing ingested Saccharomyces cerevisiae is inhibited by polyanions previously incorporated in the medium. In contrast, this fusion process can be accelerated by chloroquine and some other secondary and tertiary amines; and these compounds can reverse the inhibition induced by a polyanion. The non-fusion pattern usually associated with intracellular Mycobacterium tuberculosis can also be reversed by chloroquine. Our observations offer a possible new approach to the study of subcellular membrane fusion, and of factors influencing the course of experimental intracellular infections. Summary.
It is still uncertain what role the lysosomal system plays in determining whether microorganisms ingested by macrophages survive or die. Many species of microorganism that are phagocytosed by macrophages are The authors wish to thank Miss Naoko Hieda for her rapidly exposed to the contents of the lysotechnical assistance, Dr T. Shiomi for discussion, and somes as a result of phagosome-lysosome Mr R. L. Simmons for improving English. This work was supported in part by grants-in-aid from the Min- fusion (P-LF). Some pathogens, notably istry of Education, Science and Culture, Japan. virulent Mycobacterium tuberculosis [I] and living Toxoplasma gondii [2] are excepReferences tions; in cultured normal macrophages P1. Schmid, W, Mutat res 3 1 (1975) 9. 2. Ege, T & Ringer& N R, Exp cell res 87 (1974) LF is inhibited after phagocytosis. 378. It seemed possible that evasion of con3. Johnson, R T, Mullinger, A M & Skaer, R J, Proc roy sot B 189(1975) 591. tact with lysosomal contents might assist in4. Shiomi, T & Sato, K, Exp cell res 100(1976) 297. tracellular survival of these microorganS. Deitch, A D, Introd quant cytochem 1 (1966) 327. isms. Attachment of antibody to them beReceived January 18, 1978 fore their ingestion reversed the non-fusion Revised version received March 8, 1978 Accepted March 13, 1978 patterns, and led to killing and digestion of Toxoplasma [3, 41, but the multiplication of tubercle bacilli was uninhibited [5]. We sought other ways of controlling P-LF, so as to learn more about its mechanism and its functions in intracellular infections. Sev-
Preliminary
notes
487
Table 1. Proportion of yeast-containing phagosomes showing fusion with AO-labelled lysosomes in macrophages exposed to chloroquine diphosphate (10 pg ml-’ for 30 min) following ingestion of irradiated yeasts
Untreated (controls) Chloroquine-treated
% with intraphagosomal orange “rims””
% with intraphagosomal coloured yeastsb
Total % phagosomes fused
60 20
10 75
70 95
n Confluent rims of A0 fluorescence within the phagosomes and surrounding the enclosed yeasts (i.e. early stage of fusion). * Penetration of the yeasts by the A0 (i.e. later stage of fusion).
era1polyanionic substances were found that were taken up from the culture medium by endocytosis and accumulated in secondary lysosomes, and which inhibited the fusion of the latter (though probably not of primary lysosomes) with phagosomes containing yeast cells (which normally promote PLF [6, 71). We now report, in contrast, enhancement of the P-LF response by the antimalarial drug chloroquine, and preliminary results with some other secondary and tertiary amines. Added to cell culture medium, chloroquine enters the cell, probably by diffusion, and is rapidly concentrated in the lysosomes [g-l 11.We have assessedthe extent of P-LF by dark-field vital fluorescence microscopy and by electron microscopy r&71.
Materials
and Methods
Fluorescence microscopy (FM): celeration of P-LF (yeast target).
Assessment
of ac-
Normal mouse peritoneal macrophages, established as monolayers in Chang medium on coverslips in Leighton tubes [ 1, 5, 121for 1-2 weeks, were given a change of medium, and used l-5 days later. The monolayers were washed with balanced salt solution (BSS) and exposed at 37°C to acridine orange (AO, 5 WEml-‘) in BSS for 10 min (to prelabel secondary lysoiomes) [6, 71. The cells were then washed and incubated at 37°C with a suspension in BSS of commercial baker’s yeast, Saccharomyces cerevisiae (5~ lo8 yeast cells ml-l). used either fresh or after exposure to 150 krad gamma radiation (giving a survival c 1%). After 15 or 30 min the macrophages were washed and exposed at 37°C in BSS to chloroquine diphosphate (ICI Pharmaceuticals), usually 10 pg ml-’ (20 PM) for 30 or 45 min;
this dose was well tolerated by the cells (l-2 weeks). Control tubes without the drug were incubated in parallel. Then, after a final wash the coverslips were examined under blue-violet light (within 60 min after completion of the yeast ingestion). The extent of PLF was judged by observing passage of the fluorescent lysosomal marker (AO) into yeast-containing phagosomes [6, 71. The results were similar if the macrophages were exposed to the drug before the A0 labelling and subsequent ingestion of yeasts, but were less consistent. FM: Assessment of reversal qf inhibition of P-LF (yeast target). The procedure wasas above except that
poly-n-glutamic acid (PGA) (sodium salt, 100pg ml-‘) had been incorporated in fresh culture medium for 5 days; the monolayers were then washed, exposed in turn to AO, live or irradiated yeasts, and chloroquine. Change of the sequence from PGA-AG-veasts-chloroquine-to PGA-chloroquine-AG-yeasts did not affect results. Electron of inhibition
microscopy (EM): Assessment of reversal of P-LF (yeast target). The macrophage
monolayers were exposed to ferritin (10 mg ml-‘) for 3 h (to prelabel secondary lysosomes) [ 1, fi. The next day, PGA (100 ~g ml-‘) was added in fresh medium and the tubes were incubated for 5 days at 37 “C. The cultures were washed, irradiated veasts added. and the tubes reincubated for 30 min. After further ‘washing of cells, chloroquine diphosphate (10 +g ml-‘) was added to half the tubes. The macrophages were fixed after 60 min of further incubation [ 1, 131. EM: Reversal of inhibition (Mycobacterium tuberculosis target). Ferritin-prelabelled macrophages were
exposed at 37°C for 90 min to a suspension of strain H37Rv [ 11. After washing, chloroquine diphosphate (20 pg ml-‘) was added to half the tubes, and all were reincubated for 45 min. The cultures were again washed, incubated in fresh BSS for a further 60 min. and then fixed.
Results and Discussion
Acceleration of the phagosome-lysosome fusion process, judged by FM with yeasts as target, occurred in chloroquine-treated Erp Cd/ Rrs i 14 f 1978)
488
Preliminary
notes
Table 2. Proportion
of yeast-containing phagosomes showing fusion with AO-labelled lysosomes in macrophages pretreatedfor 5 days with poly-o-glutamic acid (100 pg ml-‘); then permitted to ingest irradiated yeasts; andfinally exposed to chloroquine diphosphate (10 p.g ml-‘for 30 min) PGA treatment
Chloroquine treatment
% with intraphagosomal orange “rims””
% with intraphagosomal coloured yeasts*
Total % phagosomes fused
No Yes Yes
No No Yes
55 0 50
15 0 15
70 0 65
a,* See table I.
macrophages compared with controls (table 1). Chloroquine was found also to reverse the inhibition of P-LF induced in macrophages pretreated with a polyanion, the results of a typical experiment being shown in table 2. If the PGA concentration was raised to 1.0 mg ml-’ the reversal was partial, but if the chloroquine concentration was also raised to 20 pg ml-’ for 45 min, reversal was fully restored. The reversal by chloro&ine of the PGAinduced inhibition of the P-LF response to yeasts was also shown by EM. In thin sections of cells treated with PGA alone, ferritin label was not seen in any of 200 phagosomes counted, 90% of which were of the “loose” variety [6, 71. In the macrophages treated with PGA and chloroquine, ferritin was present in 20% of 250 phagosomes counted. These differences, indicating partial reversal of inhibition, were significant (P~0.01). All ferritin-containing phagosomes were of the “tight” variety: where non-fusion had not been reversed (80%), the phagosome membranes were “tight” or “loose” in equal proportions. ‘The effect of chloroquine on the P-LF response to infection with M. tuberculosis was also examined by electron microscopy. The typical non-fusion pattern of lysosomal response to virulent M. tuberculosis [ 11was Exp Cd RPS I14 (1978)
almost completely reversed by the drug. Ferritin was seen in only about one-fifth .of phagosomes containing intact bacilli in the control macrophages, but in three-quarters of them in chloroquine-treated cells. At this dose the drug characteristically [14] induced many autophagic vacuoles, which frequently contained ferritin as well as debris and were amalgamated with the bacillus-containing phagolysosomes. (After exposure to the more usual concentration of 10 pg ml-’ (see above), autophagic vacuoles were inconspicuous.) S. cerevisiae, exposed in vitro at 37°C for 30 min to a high concentration of chloroquine diphosphate (1 .O mg ml-‘), binds the drug (demonstrable by fluorescence) and is killed. If yeasts so treated, and washed free of unbound drug, are ingested by normal macrophages there is enhancement of PLF; and if ingested by PGA-pretreated macrophages the usual inhibition of P-LF observed after ingestion of normal yeasts by PGA-treated cells is absent-both situations produced with normal yeasts only after treatment of these macrophages with free chloroquine. During the exposure of macrophages (3U5 min) to chloroquine after they have ingested normal yeasts the drug will begin to enter the yeast-containing phagosomes along with other lysosomal
Preliminary
contents. The question arises, therefore, whether both the enhancement of P-LF in normal macrophages by chloroquine and its ability to reverse the inhibition of P-LF in PGA-pretreated macrophages might be due to the presence of chloroquine bound to killed yeasts within the phagosomes and not to this drug accumulated in the lysosomes and acting at that site. However, in experiments where PGA-pretreated monolayers had ingested normal live yeasts, and were then exposed to chloroquine (10 pg ml-’ for 30 min), culture counts of the intracellular yeasts (after disruption of the macrophages by gentle ultrasonic vibration) revealed little or no loss of viability over this limited period. Furthermore, a chloroquine-resistant strain of S. cerevisiae behaved altogether like chloroquine-sensitive yeasts: PLF in normal macrophages was enhanced, and its inhibition in PGA-pretreated macrophages was reversed, only when the macrophages were exposed additionally to free chloroquine in the usual way. This strain did not bind chloroquine after exposure for 30 min in vitro to 1.0 mg ml-‘, nor was it killed by this exposure. We conclude that both the enhancement and the inhibitionreversal effects on P-LF by chloroquine are direct effects upon the cell, rather than because the drug becomes attached to the intraphagosomal yeast. The reversal by chloroquine of PGA-induced inhibition of P-LF might result from direct ionic interaction of chloroquine with the polyanion in the secondary lysosomes: however, chloroquine on its own is able to enhance fusion above normal levels. We conclude that the compounds act competitively on the cells. Some other tertiary and secondary amines have been similarly examined for their ability to accelerate the P-LF response to ingestion of yeasts in normal macro-
notes
489
phages, and to reverse its inhibition induced in polyanion (PGA)-pretreated macrophages. The following additional compounds were found to be active in both respects: quinacrine dimethane sulphonate, 3-quinuclidinyl benzilate, atropine sulphate, tetracaine hydrochloride, tri-n-butylamine and di-n-butylamine hydrochlorides (quinacrine and chloroquine being the most active on a molar basis). Triethylamine hydrochloride was weakly active, and N,Ndimethylaniline inactive. In separate experiments chloroquine, tetracaine and tributylamine have been tested for their effect on the progress of a tuberculous infection within the cultured macrophages. Each of these compounds inhibited or suppressed intracellular bacillary multiplication. The identification of some chemical effector agents able to control P-LF experimentally may offer another approach to the study of the mechanism of this membrane fusion process, as well as to the possibility of influencing the course of a pathogenic infection by this type of manipulation. We thank Drs J. A. Armstrong and P. Draper for advice and encouragement throughout this investigation; Drs G. H. Beaven, R. H. Gigg, N. J. M. Birdsall and D. C. Warhurst for technical suggestions; and Dr D. H. Williamson for the chloroquine-resistant strain of S. cerevisiae.
References 1. Armstrong, J A & Hart, P D’Arcy, J exp med 134 (1971) 713. 2. Jones, T C & Hirsch, J G, J exp med 136 (1972) 1173. 3. Jones, T C, Jreticuloendothelial sot 15(1974)439. 4. Jones, T C, Len, L & Hirsch, J G, J exp med 141 (1975) 466. 5. Armstrong, J A & Hart, P D’Arcy, J exp med 142 (1975) 1. 6. Hart, P D’Arcy & Young, M R, Nature 256 (1975) 47. 7. Goren, M B, Hart, P D’Arcy, Young, M R & Armstrong, J A, Proc natl acad sci US 73 (1976) 2510. 8. Allison, A C &Young, M R, Life sci 3 (1964) 1407. Exp Cell Res I14 (1978)
490
Preliminary notes
9. de Duve, C, de Barsy, T, Poole, B, Trouet, A, Tulkens, P & Van Hoof, F, Biochem pharmacol23 (1974) 2495. IO. Wibo, M & Poole, B, J cell bio163 (1974) 430. 11. Reijngoud, D J & Tager, J M, FEBS lett 64 (1976) 231. 12. Hart, P D’Arcy, Science 162(1968) 686.
Exp Cell Res 114 (1978)
13. Hirsch, J G & Fedorko, M E, J cell biol 38 (1968) 615. 14. Fedorko, M E, Hirsch, J G & Cohn. 2 A, J cell bio138 (1%8) 377. Received January 20, 1978 Accepted February 16, 1978
