INFECTION AND IMMUNITY, July 1979, p. 463466

Vol. 25, No. 1


Cytochalasin B Does Not Inhibit Ingestion of Chlamydia psittaci by Mouse Fibroblasts (L Cells) and Mouse Peritoneal Macrophages WILLIAM W. GREGORY,t GERALD I. BYRNE,4 MOREY GARDNER,§ AND JAMES W. MOULDER* Department of Microbiology, University of Chicago, Chicago, Illinois 60637 Received for publication 12 February 1979

Cytochalasin B did not inhibit ingestion of Chlamydia psittaci by either mouse fibroblasts (L cells) or mouse peritoneal macrophages in concentrations that produced distinctive morphological changes and inhibited phagocytosis of polystyrene latex beads and Escherichia coli K-12. The fungal metabolite cytochalasin B (15) is a powerful inhibitor of phagocytosis (4, 11), probably because it prevents the gelation of actin by actin-binding protein and thereby specifically and reversibly inhibits the functioning of microfilaments (14). Even the active, energyrequiring entry of typhus rickettsiae (16) and malarial parasites (10) into host cells is interfered with by cytochalasin B. Chlamydia psittaci is taken into mouse fibroblasts (L cells) (5, 8) and mouse macrophages (17) by a mechanism

morphologically indistinguishable from classical phagocytosis, and the energy for its endocytosis comes from host cells (3, 5) and not from the chlamydiae, which do not generate metabolically useful energy (6). We have found that the ingestion of C. psittaci by either L cells or mouse macrophages is unusual in that it is not inhibited by cytochalasin B. Multiplication of chlamydiae in cells treated with cytochalasin B has already been described (7, 9, 12, 13) under conditions that do not allow quantitative measurement of ingestion. L cells and the 6BC strain of Chlamydia psittaci were propagated by the method of Hatch (7). Unelicited macrophages were obtained from peritoneal washings of 6- to 8-week-old, pathogen-free CD-1 mice (Charles River Laboratories). Macrophage monolayers were maintained in modified Eagle medium with nonessential amino acids, 0.1% sodium bicarbonate (5% C0295% air), 200 ,tg of streptomycin sulfate per ml, and 10% heat-inactivated fetal calf serum. Mact Present address: Department of Microbiology, University of Rochester Medical Center, Rochester, NY 14642. t Present address: Division of Infectious Diseases, Department of Medicine, New York Hospital-Cornell Medical Center, New York, NY 10021. § Present address: Division of Infectious Diseases, Department of Medicine, Jewish Hospital of St. Louis, St. Louis, MO 63110.

rophage populations were rinsed free of nonadherent cells 24 h after they had been plated out in 25-cm2 plastic tissue culture flasks at a density of 40,000 cells per cm2. A full description of these procedures will be published elsewhere (M. Gardner, manuscript in preparation). Escherichia coli K-12 labeled with 14C amino acids was prepared as described previously (3). Cytochalasin B (Aldrich Chemical Co.) was dissolved in dimethyl sulfoxide and diluted in growth medium just before use. Scanning electron microscopy was performed by the method of Byrne (2). Phagocytosis of 1-Itm-diameter polystyrene latex spheres was determined by direct counting with phase-contrast or fluorescence microscopy (1). Phagocytosis of 14C-labeled E. coli K-12 was determined by modification of a previously published procedure for L cells (1, 3). Phagocytosis of unlabeled C. psittaci by L cells and macrophages was followed by the method of Byrne (1, 2), which is based on the 50% infectious unit titration procedure of Hatch (7). In all three assays, the extent of phagocytosis in the presence and absence of cytochalasin B was determined, and the results were expressed in terms of phagocytosis as percentage of control. When L cells inoculated with C. psittaci were examined by scanning electron microscopy, numerous elementary bodies were seen attached to host cell surfaces. Some of them appeared to be partially enveloped by evaginaced plasma membrane and were probably in the initial stages of endocytosis. Figure 1A shows an elementary body of C. psittaci circumscribed by an evagination of the L-cell plasma membrane. Figure 1B may represent a later stage of ingestion in which pseudopodia extend to engulf the chlamydial cell. If cytochalasin B inhibits the ingestion of C. psittaci by mouse fibroblasts and mouse mac-



FIG. 1. Scanning electron micrographs of elementary bodies of Chlamydia psittaci attached to L cells. (A) A single chlamydial cell (arrow) circumscribed by an evagination of the plasma membrane of the L cell. The bar represents 0.5 ,um. x 75,000. (B) Two elementary bodies (arrows) with L-cell pseudopodia extended around them. The bar represents 0.5 ,um.




with microfibrils extending from the leading edge. The appearance of the cell in Fig. 2B is typical of L cells treated for 60 min at 370C with 2 x 10-6 M cytochalasin B, the minimal cytopathic concentration for these host cells. Note the rounded dendritic processes extending from the cell cortex and the sparse distribution of microvilli. By 24 h, L cells treated with 2 x 106 M cytochalasin B were binucleate. They excluded trypan blue, but they did not divide. Untreated macrophages spread on glass substrates were flat and elongated with microfibrils projecting from the cell margins (Fig. 20). When macrophages were exposed to as little as 1 x 106 M cytochalasin B for 60 min at 370C, their cell margins retreated toward cell centers and left occasional processes stretching outward toward points of substrate attachment (Fig. 2D). Morphological changes induced by minimal cytopathic doses of cytochalasin B were reversed by washing and suspending in fresh medium, but with higher drug concentrations, increasing fractions of both L-cell and macrophage populations ceased to exclude trypan blue and did not regain normal morphology in fresh medium. The effect of up to 10 times the minimal cytopathic concentration of cytochalasin B on the ingestion and subsequent intracellular development of C. psittaci in L cells and macrophages was measured in terms of inclusion formation after inoculation with one L-cell 50% infectious unit per host cell. For each kind of host cell, a second ingestible particle was added to the assay system, 1-tIm-diameter polystyrene latex spheres for L cells and 14C-labeled E. coli for macrophages. At the minimum cytopathic level, cytochalasin B did not inhibit the phagocytosis of any of the three particulates by either host cell, suggesting that phagocytosis is not among the cellular activities most susceptible to derangement by this drug. However, higher concentrations of cytochalasin B differentially affected phagocytosis of C. psittaci and polystyrene latex beads by L cells (Fig. 3A). The uptake and intracellular development of C. psittaci was not affected, but the uptake of latex beads was progressively inhibited by increasing concentrations of the drug to a maximum of almost 75% at 2 x 10'5 M. A comparable differential effect of cytochalasin B on the phagocytosis of C. psittaci and 14C-labeled Escherichia coli by mouse macrophages was also observed (Fig. 3B). The* highest concentration of -cytochalasin B tested (2 x 10-5 M) did not reduce the percentage of host cells in which chlamydial inclusions developed, but it reduced the association of labeled E. coli with macrophages by more than

rophages, then such inhibition should be evident at a concentration of the drug equal to or somewhat greater than that just sufficient to produce distinctive morphological alterations in these host cells (minimal cytopathic concentration). Figure 2A shows an uninfected L cell spread on a glass substrate and observed by scanning electron microscopy. It has a regular margin with abundant microvilli over the nuclear area and 70%.

VOL. 25, 1979



FIG. 2. Scanning electron micrographs of uninfected L cells and mouse peritoneal macrophages spread on glass substrates. (A) An untreated L cell. (B) An L cell treated with 2 x 10-6 M cytochalasin B for 60 min at 37C. (C) An untreated mouseperitoneal macrophage. (D) A macrophage treated with 1 x 10-6 M cytochalasin B for 60 min at 37°C. The bar in A represents 10 pvm. x2,000.

In conclusion, the entry of C. psittaci into either professional phagocytes (mouse macrophages) or nonprofessional phagocytes (mouse fibroblasts) appears to take place by a mechanism that is less dependent on those functions of microfilaments that are disrupted by cytochalasin B than are the mechanisms whereby polystyrene latex spheres and E. coli are ingested. This conclusion does not exclude the

possibility that cytoplasmic contractile elements may contribute to the highly efficient mechanism of entry that chlamydiae use to infect host cells, but they do suggest that there may be something different about this contribution. The observation that pinocytosis, unlike phagocytosis, is frequently insusceptible to inhibition by cytochalasin B may offer a clue as to the nature of the difference (4, 11).





This investigation was supported by Public Health Service research grant AI-13175 and postdoctoral research fellowship grant AI-05405, both from the National Institute of Allergy and Infectious Diseases, and by Public Health Service training grant GM-603 from the National Institute of General Medical Sciences.


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I. 1976. Requirements for ingestion of Chiamydia psittaci by mouse fibroblasts (L cells). Infect. Immun. 14:645-651. 2. Byrne, G. I. 1978. Kinetics of phagocytosis of Chlamydia psittaci by mouse fibroblasts (L cells): separation of the attachment and ingestion stages. Infect. Immun. 19: 607-612. 3. Byrne, G. I., and J. W. Moulder. 1978. Parasite-specified phagocytosis of Chlamydia psittaci and Chlamydia trachomatis by L and HeLa cells. Infect. Immun. 19:

1. Byrne, G.





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FIG. 3. Effect of cytochalasin B on uptake of C. psittaci, polystyrene latex spheres, and E. coli by L cells and mouse peritoneal macrophages. Monolayers were incubated with the drug for 90 min at 370C, the particles to be phagocytized were added, and uptake was determined after an additional 60 min at 370C. For each concentration of cytochalasin B tested, uptake as percentage of control was calculated by comparing the uptake in the presence of cytochalasin B dissolved in dimethyl sulfoxide growth medium with the uptake in the presence of dimethyl sulfoxide growth medium alone. (A) Effect of cytochalasin B on uptake of C. psittaci and 1-jim-diameter polystyrene latex spheres by L cells. Chlamydial ingestion was measured in terms of the percent inclusion-bearing cells after inoculation of one L-cell 50% infectious dose per host cell. In the absence of cytochalasin B, 49% of the L cells developed one or more chlamydial inclusions. Uptake of latex beads was estimated by direct counting after addition of 1,000 beads per L cell. In the absence of the drug, the mean uptake per L cell was 7.8 latex spheres. Symbols: 0, C. psittaci; 0, polystyrene latex spheres. (B) Effect of cytochalasin B on uptake of C. psittaci and "4C-labeled E. coli by mouse peritoneal macrophages. Chlamydial ingestion was measured as in A. In the absence of the drug, one L-cell 50% infectious dose infected 39% of the macrophage population (0. 71 50% infectious dose per macrophage). Phagocytosis of E. coli was estimated by measuring the 14C counts associated with macrophages that had been incubated with 10 4Clabeled E. coli cells per macrophage. In the absence of the drug, the mean radioactivity associated with each macrophage was equivalent to 2.9 E. coli cells. Symbols: 0, C. psittaci; 0, E. coli. We thank Willie M. Conway for technical assistance. The scanning electron micrographs were taken in the Microscope User's Laboratory of the University of Chicago, which is supported by a grant from the Biotechnical Resources Branch of the National Institutes of Health.

598-606. 4. Davies, P., and A. C. Allison. 1978. Effects of cytochalasin B on endocytosis and exocytosis, pp. 143-160. In S. W. Tanenbaum (ed.), Cytochalasins-biochemical and cell biological aspects. Elsevier/North Holland Biomedical Press, Amsterdam. 5. Friis, R. R. 1972. Interaction of L cells and Chlamydia psittaci: entry of the parasite and host responses to its development. J. Bacteriol. 110:706-721. 6. Hatch, T. P. 1975. Utilization of L-cell nucleoside triphosphates by Chlamydia psittaci for host ribonucleic acid synthesis. J. Bacteriol. 122:393-400. 7. Hatch, T. P. 1975. Competition between Chlamydia psittaci and L cells for host isoleucine pools: a limiting factor in chlamydial multiplication. Infect. Immun. 12:

211-220. 8. Higashi, N. 1965. Electron microscopic studies on the mode of reproduction of trachoma virus and psittacosis virus in cell cultures. Exp. Mol. Pathol. 4:24-39. 9. Kuo, C.-C. 1978. Cultures of Chlamydia trachomatis in mouse peritoneal macrophages: factors affecting organism growth. Infect. Immun. 20:439-445. 10. Miller, L. H., M. Aikawa, J. G. Johnson, and T. Shiroishi. 1979. Interaction between cytochalasin Btreated malarial parasites and erythrocytes. J. Exp. Med. 149:172-184. 11. Silverstein, S. C., R. M. Steinman, and Z. A. Cohn. 1977. Endocytosis. Annu. Rev. Biochem. 46:669-722. 12. Sompolinsky, D., and S. Richmond. 1974. Growth of Chlamydia trachomatis in McCoy cells treated with cytochalasin B. Appl. Microbiol. 28:912-914. 13. Stirling, P., and S. Richmond. 1977. The developmental cycle of Chlamydia trachomatis in McCoy cells treated with cytochalasin B. J. Gen. Microbiol. 100:31-42. 14. Stossel, T. P., and J. H. Hartwig. 1976. Interactions of actin, myosin, and a new actin-binding protein of rabbit pulmonary macrophages. II. Role in cytoplasmic movement and phagocytosis. J. Cell. Biol. 68:602-619. 15. Tanenbaum, S. W. 1978. Microbiological, preparative and analytical aspects of cytochalasin production, p. 114. In S. W. Tanenbaum (ed.), Cytochalasin-biochemical and cell biological aspects. Elsevier/North Holland Biomedical Press, Amsterdam. 16. Walker, T. S., and H. H. Winkler. 1978. Penetration of cultured mouse fibroblasts (L cells) by Rickettsia prowazeki. Infect. Immun. 22:200-208. 17. Wyrick, P. B., and E. A. Brownridge. 1978. Growth of Chiamydiapsittaci in macrophages. Infect. Immun. 19: 1054-1060.

Cytochalasin B does not inhibit ingestion of Chlamydia psittaci by mouse fibroblasts (L cells) and mouse peritoneal macrophages.

INFECTION AND IMMUNITY, July 1979, p. 463466 Vol. 25, No. 1 0019-9567/79/07-0463/04$02.00/0 Cytochalasin B Does Not Inhibit Ingestion of Chlamydia...
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