INFECTION
AND
Vol. 14, No. 3 Printed in U.S.A.
IMMUNrrY, Sept. 1976, p. 645-651
Copyright 0 1976 American Society for Microbiology
Requirements for Ingestion of Chlamydia psittaci by Mouse Fibroblasts (L Cells) GERALD I. BYRNE
Department of Microbiology, University of Chicago, Chicago, Illinois 60637
Received for publication 23 March 1976
Ingestion of "4C-amino acid-labeled Chlamydia psittaci (6BC) by mouse fibroblasts (L cells) was inhibited when the host cells were incubated for 30 min at 37°C in Earle salts containing 10 ,ug of crystalline trypsin per ml. Tryptic digestion also inhibited the ingestion of 1-,um polystyrene latex beads. Trypsintreated L cells almost completely recovered their ability to ingest chlamydiae after 4 h at 370C in medium 199 with 5% fetal calf serum. Cycloheximide (10 ,ug/ ml) blocked this recovery. Heating 14C-amino acid-labeled C. psittaci for 3 min at 60°C inhibited its ingestion by L cells, whereas inactivating it with ultraviolet light was without effect on the ingestion rate. These results show that efficient ingestion of C. psittaci by L cells involves trypsin-labile sites on the host and heat-sensitive sites on the parasite. The failure of excess unlabeled infectious C. psittaci to promote the ingestion of "4C-labeled heat-inactivated chlamydiae suggests that direct interaction between these two sites must occur for uptake to proceed normally. The genus Chlamydia is a group of obligate intracellular procaryotes that infect a wide variety of avian and mammalian cells (18, 19). In man, they cause trachoma, inclusion conjunctivitis, lymphogranuloma venereum, psittacosis, and possibly nongonococcal urethritis (9). In both natural infections and cell cultures, chlamydiae readily gain entry into host cells that are not actively phagocytic, and the actual mechanism of their uptake has remained obscure (8, 13, 14, 21). The objective of this investigation was to learn more about the surface interactions between Chlamydia psittaci and mouse fibroblasts (L cells) that must precede ingestion of the parasite by the host cell.
ln 0.5 ln decimal fraction uninfected cells x decimal dilution = ID50 for particular number of L cells used Preparation of trypsin-treated L cells. L cells were collected from suspension by centrifugation at 500 x g for 10 min and resuspended in Earle balanced salt solution (BSS) (7) at a density of 106 cells/ ml. Measurement of the effect of treating the L cells with trypsin with respect to the rate at which they ingested C. psittaci was performed by distributing the L-cell suspensions in 5-ml volumes to 25-ml Erlenmeyer flasks that had been equilibrated in 5% CO2-95% air. Different volumes of a filter-sterilized solution of crystalline trypsin (Worthington Biochemicals) in the phosphate-buffered saline of Dulbecco and Vogt (PBS) (6) were added to duplicate flasks, and the total volume of the L-cell suspensions was adjusted to 5.5 ml with PBS. The flasks were closed with silicone rubber stoppers and shaken at 370C for 30 min at 125 strokes/min. The trypsin-treated L cells were then collected by centrifugation and resuspended to their original density in growth medium.
MATERIALS AND METHODS Growth of L cells and chlamydiae. The methods used for growing mouse fibroblasts (L cells) in suspension culture, for infecting them with C. psittaci (6BC), and for preparing harvests of C. psittaci have recently been described (10). Medium 199 supplemented with 5% heat-inactivated fetal calf serum and containing 0.1% sodium bicarbonate and 200 ,ig of streptomycin sulfate per ml was used for maintaining L cells and for propagating chlamydiae. It will be referred to simply as "medium" or "growth medium." L cells were counted in a Coulter counter, and their viability was determined by the trypan blue exclusion test (16, 17). Hatch's method (10) for titrating chlamydial infectivity was used.,It consists of determining the dose of chlamydiae needed for infecting 50% of a population of L cells of known density (IDso) according to the relation:
Preparation of "4C-labeled C. psittaci. To prepare
radioisotopically labeled chlamydial cells, the infection was carried out in medium that had been supplemented with 1 ,uCi of a "4C-L-amino acid mixture (New England Nuclear Corp.; average specific activity, 259 mCi/mmol) per ml. Incorporation of the radioisotope specifically into chlamydial protein was facilitated by addition of 2 pg of cycloheximide per ml (The Upjohn Co. ) to inhibit synthesis ofhost protein (1). The "4C-labeled C. psittaci was purified by 645
646
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INFECT. IMMUN.
enzyme digestion and differential centrifugation ac- cells infected was determined as in an ID50 titration. cording to Hatch (10). When freshly purified C. psit- Although this procedure measures uptake plus detaci was used for infection at a multiplicity of 1 to 10 velopment to a microscopically visible inclusion, it ID50 per host cell, more than 80% of the precipitable gives a reasonable reflection of the rate of chlam14C label became associated with L cells after 2 h at ydial ingestion when 1 ID50 per cell is used. Only 370C. This represented essentially all of the infec- 50% of the L cells are infected at a maximum, and tious chlamydiae present as determined by retitra- most of the host cells contain only a single inclusion. tion of the supernatant fluid after a single centrifu- Thus, multiply infected cells are not a complicating gation at 500 x g for 10 min. Labled C. psittaci factor, and one infected cell comes close to reprepreparations equivalent to 5 x 106 L-cell ID50 doses senting one ingestion event. Quantitating ingestion had counts in the range of 400 to 800 cpm. by this method also obviates the problem of distinInactivation of C. psittaci with heat and ultravi- guishing between external attachment and true upolet light. Chlamydiae that had been grown in unla- take, since only those chlamydiae that are actually beled or in '4C--amino acid-labeled medium were taken into the L cells are able to replicate and form harvested from infected L cells, purified as just de- inclusions. Infected cell counts were made by somescribed, and suspended in PBS. Two milliliters of one who was not aware of the origin of the samples the suspension was then placed in a plastic petri counted. dish with a surface area of 28 cm2 and inactivated Measurement of the ingestion of polystyrene lawith ultraviolet light (3-min exposure to a 30-W tex spheres. When the ingestion of polystyrene latex General Electric germicidal lamp at'a distance of 76 spheres (Dow Diagnostics; 1-,um diameter) was to be cm). A 2-ml portion of the PBS suspension was measured, L cells were suspended in growth meplaced in a 13-mm-diameter tube and shaken for 3 dium to a density of 106 cells/ml and distributed into min in a 60°C water bath and then cooled in an ice flasks as already described. Latex spheres were then bath. Such heating did not macroscopically or mi- added in a ratio of 500 spheres for every L cell. croscopically clump the chlamydial cells and did not Samples were shaken at 370C for 60 min and washed alter their sedimentation properties. three times with growth medium to remove unassoMeasurement of the ingestion of 14C-labeled chla- ciated spheres from the cells. The L cells were then mydiae. For experiments in which the ingestion of diluted to a density of 0.25 x 106 cells/ml in growth 14C-labeled chlamydiae was to be measured, appro- medium containing 10% heat-inactivated fetal calf priately treated L cells were suspended to a density serum and plated out in plastic tissue culture flasks of 106 cells/ml in growth medium. The cell suspen- at a density of 5 x 104 cells/cm2. After 4 h at 370C, sion was then distributed in 5-ml portions to equili- the monolayers were washed three times with PBS brated 25-ml flasks, and 1 ID50 per L cell of a freshly and fixed in absolute ethanol. The number of titered 14C-labeled C. psittaci suspension was added spheres ingested by 200 L cells was then counted by to each flask. The flasks were shaken at 370C for 30 phase-contrast microscopy. Sphere counts were to 120 min. The L cell-chlamydiae mixtures were made by someone who did not known the nature of then centrifuged at 4°C for 5 min at 300 x g. This the samples counted. provided sufficient. force to sediment the L cells and Demonstration of the regeneration of trypsinL-cell-associated chlamydiae, but not enough force labile sites on L cells. These experiments were done to sediment free, unattached chlamydiae. To be sure by treating L cells with 10 ,ug of trypsin per ml as that all the unassociated chlamydiae had been described in a earlier section. The cells were centriwashed from the host cells, the L-cell pellet was fuged, resuspended to a density of 106 cells per ml of resuspended in ice-cold growth medium and recen- growth medium, and transferred to Erlenmeyer trifuged. This procedure was repeated an additional flasks in 5-ml volumes. Another part of the same Ltime. Monitoring the radioactivity in the successive cell population was carried through the same masupernatant fluids showed that all the free chlamy- nipulations without the addition of trypsin, and the diae were removed by three washes. After the third same number of flasks was prepared for these uncentrifugation, the L cells were resuspended in 2 ml treated L cells. Four untreated and four trypsinof 5% trichloroacetic acid, and the precipitable treated flasks were set up for each recovery period to counts were collected on membrane filters (Milli- be tested. Cycloheximide at a final concentration of pore Corp.) by a previously published procedure (1). 10 Ag/ml was added to two of each set of four flasks. Samples were counted in 5 ml of a toluene-based The L-cell suspensions were shaken at 370C; after 0, scintillant in a Packard Tri-Carb liquid scintillation 30, 60, 120, and 240 min, 1 ID50 per L cell of either unlabeled or 14C-labeled C. psittaci was added, and spectrometer. Measurement of the ingestion of unlabeled shaking was continued for another 60 min. At apchlamydiae. Infection with 1 IDO per host cell, propriate times, samples were removed from the incubation, and washing the infected L cells by cen- shaker, and the uptake of labeled or unlabeled C. trifugation were carried out as just described. The psittaci was determined as described earlier. final L-cell pellet was resuspended to original denEffect of excess unlabeled, infectious C. psittaci sity in growth medium containing 10% heat-inacti- on the ingestion of '4C-labeled, heat-killed chlamyvated fetal calf serum and was diluted fourfold in diae. In these experiments, 5 or 50 ID50 per L cell of the same medium, and 1-ml volumes were added to unlabeled, purified, infectious chlamydiae was duplicate cover slip-containing Leighton tubes (25), mixed with 1 ID50 of 14C-labeled, purified, heat-inacwhich were incubated for 18 to 24 h at 370C in 5% tivated C. psittaci, and samples were added to dupliC02-95% air. They were then stained with Giemsa cate flasks containing 5 ml of an L-cell suspension at stain according to Hatch (10). The percentage of L 106 cells/ml. Ingestion of the 14C-labeled C. psittaci
VOL. 14, 1976
CHLAMYDIAL INGESTION BY L CELLS
was followed by the procedure already described. Samples that contained 1 ID,0 of infectious, 14Clabeled chlamydiae and samples that contained the 14C-labeled, heat-killed chlamydiae alone were included in each experiment.
RESULTS Inhibition of the ingestion of C. psittaci by tryptic digestion of L cells. When L cells were suspended in Earle balanced salt solution, treated with trypsin for 30 min at 370C, and resuspended in growth medium, as little as 1 ,ug of trypsin per ml inhibited the ingestion of infectious C. psittaci (Fig. 1). A maximum inhibition of more than 80% was obtained with 10 ,ug of trypsin per ml. When the L cells were treated with trypsin while suspended in growth medium containing 5% heat-inactivated fetal calf serum, a potent inhibitor of tryptic activity, no decrease in the ability to ingest chlamydiae was observed. Recovery of the ability to ingest C. psittaci and its inhibition by cycloheximide. When the
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FIG. 2. Recovery of the ability of trypsin-treated L cells to ingest C. psittaci and inhibition of recovery by cycloheximide: use of unlabeled chlamydiae to measure ingestion. Ingestion of unlabeled chlamydiae was followed by the procedure given in Materials and Methods. One hundred percent uptake was the ingestion ofunlabeled infectious C. psittaci found at 1 h in populations ofcells that were not treated with trypsin or with cycloheximide and were not preincubated in recovery medium. Each point is the mean ofsix determinations, and each bar represents ± 1 standard deviation. Symbols: no trypsin treatment and no cycloheximide in recovery medium (O); no trypsin treatment and 10 pg ofcycloheximide per ml in recovery medium (U); 10 pg of trypsin per ml for 30 min at 370C and no cycloheximide in recovery medium (0); trypsin treatment and cycloheximide in recovery medium (O).
observed that Rickettsia tsutsugamushi easily entered mouse lymphoblasts and fibroblasts (L cells) but that, under similar conditions, Escherichia coli and Staphylococcus aureus got into these host cells only with great difficulty. L cells also ingest C. psittaci much more efficiently than they do most other microorganisms. In experiments not described here, E. coli was labeled with "4C-amino acids to such an extent that ingestion of less than 1 bacterial cell per L cell could have been detected. Yet when L cells were exposed to labeled E. coli in a ratio of 1,000 bacterial cells per L cell, the L cell-associated label after 2 h at 370C was not significantly greater than that of the 0-h control. In contrast, when "4C-labeled C. psittaci were added to L-cell suspensions under identical conditions, nearly half of the chlamydiae were ingested when 1,000 C. psittaci were added per L cell, and essentially all of the chlamydiae were ingested when the ratio of chlamydial cells to L cells was less than 100:1. Therefore the principal objective of this investigation was to find out why C. psittaci, like many other obligate intracellular parasites, is ingested with unusual efficiency by cells that do not readily phagocytose most other microorganisms. There is little doubt that C. psittaci enters L cells by phagocytosis. Inside the host cell, the chlamydiae always lie within membrane-bound vacuoles assumed to be phagosomes (3, 8, 11), and the metabolic energy for chlamydial entry is derived from the L-cell host and not from the parasite (8, 22). The disproportionate efficiency of chlamydial ingestion by L cells may be considered an example of induced phagocytosis, a term proposed by Jones (12) to describe the entry of Toxoplasma gondii into nonprofessional phagocytes. Precise mechanisms for induced phagocytosis have not been described. The profound interference with phagocytosis of C. psittaci by L cells brought about by treating the host cells with trypsin before exposing TABLE 1. Inhibition of the ingestion ofpolystyrene
latex spheres by trypic treatment of L cellsa obligate intracellular parasites is the ability to promote their ingestion by host cells that are No. of spheres Treatment of L cells not actively phagocytic (21). Jones (12) and Racell ingested/L binovitch (24) have called these cells facultative None ..... ............. 15.3 or nonprofessional phagocytes. This property Cycloheximide, 10 lug/ml ............... 14.2 enables an obligate intracellular parasite to Trypsin, 10 ,ug/ml .................. 2.8 occupy an unexploited ecological niche (20) and Cycloheximide and trypsin ............. 2.6 to avoid the potent constitutive and inducible a L cells were treated with trypsin as described in intracellular defense mechanisms of the profes- Materials and Methods. The procedure for measursional phagocytes. Obligate intracellular pro- ing ingestion of 1-,um-diameter polystyrene latex caryotes also enter established lines of mamma- spheres was also described in Materials and Methlian cells with much greater facility than do ods. Each value represents the mean of duplicate ordinary bacteria. For example, Cohn et al. (4) determinations.
CHLAMYDIAL INGESTION BY L CELLS
VOL. 14, 1976 A
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them to the chlamydial cells shows that there are trypsin-sensitive structures on the L-cell surface essential for chlamydial uptake. Rabinovitch (23) has separated phagocytosis by macrophages into phases of attachment and ingestion. However, Friis (8) was unable to demonstrate attachment of C. psittaci to L cells in the absence of ingestion, either by using 14C-labeled chlamydiae or thin-section electron microscopy. It will be assumed that trypsin treatment interfered with an attachment of C. psittaci to L cells that is immediately followed by ingestion. This is supported by the agreement between the two methods used to assay the trypsin effect, one of which depended on attachment only (changes in L-cell-associated, 14Clabeled C. psittaci) and the other on both attachment and ingestion (changes in the percentage of L cells infected at the IDo end point). The structures destroyed by trypsin are probably recognition sites roughly analogous to the trypsin-sensitive sites on macrophages that recognize migration inhibition factor (5), complement (15), and Bacillus subtilis (2). The destruction of these sites by trypsin and the inhibition of their regeneration by cycloheximide suggests that they are protein or glycoprotein in nature. These experiments do not permit further conclusions as to the nature of the trypsin-sensitive recognition sites. The inhibition of
the ingestion of both chlamydial cells and polystyrene latex spheres by trypsin suggests that they are nonspecific. The marked reduction in the rate of phagocytosis of C. psittaci that had been heated at 60°C for 3 min is best explained by assuming that denaturation of heat-labile areas on the chlamydial cell surface destroyed its ability to induce rapid phagocytosis by L cells. The failure of inactivation with ultraviolet light to produce a comparable drop in the rate of chlamydial ingestion shows that loss of infectivity cannot in itself explain the impaired phagocytosis of heated C. psittaci. The inactivation of these putative attachment sites by brief heating at relatively low temperature suggests that they, like the recognition sites on L cells, are protein in nature. Friis (8) observed some reduction in the rate of ingestion of heated C. psittaci by L cells, but he concluded that it was not significant. However, in this investigation, repeated comparisons of the rate of phagocytosis of infectious and heat-inactivated C. psittaci showed that the heated chlamydial cells were ingested at significantly slower rates. Other experiments also demonstrated an altered reactivity of heated C. psittaci toward L cells. As described in another paper (22), very high multiplicities of heated C. psittaci had no harmful effect on L cells,
650
BYRNE
INFECT. IMMUN.
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the parasite, but further work will be necessary before any substantial conclusions can be drawn. Cohn et al. (4) studied phagocytosis of R. tsutsugamushi by mouse lymphoblasts and fibroblasts. Ingestion of rickettsiae was inhibited by inactivation either by heat or by ultraviolet light. In addition, compounds related to glutamate, which is probably oxidized by these organisms, enhanced ingestion, whereas inhibitors of glutamate oxidation depressed it. Kuo et al. (14) studied the entry of trachoma and lymphogranuloma venereum strains of Chlamydia trachomatis into HeLa 229 cells. Ingestion of trachoma strains was enhanced by pretreatment of host cells with polycations; the entry of lymphogranuloma venereum strains was inhibited. Treatment of the HeLa cells with neuraminidase reduced the uptake of trachoma strains and was without effect on lymphogranuloma venereum strains. An excess of heated trachoma cells (but not lymphogranuloma venereum cells) blocked the ingestion of infectious trachoma agent. Kuo and Grayston (13) found that heat inactivation of both trachoma and lymphogranuloma venereum strains of C. trachomatis inhibited their ingestion by HeLa 229 cells. Although it appears unlikely that a single mechanism of induced phagocytosis can explain all these diverse observations, the generalization that a heat-labile parasite component is essential for the efficient ingestion of all kinds of chlamydiae now seems to be justified. ACKNOWLEDGMENTS
MINUTES AFTER INFECTION
FIG. 4. Ingestion of infectious, heat-inactivated, and ultraviolet-inactivated 14C-amino acid-labeled C. psittaci by L cells. Methods for inactivating C. psittaci and for following the ingestion of labeled chlamydial cells are described in Materials and Methods. One hundred percent uptake was the ingestion of 14C-labeled infectious C. psittaci obtained at 3 h of incubation. Each point represents the mean of six determinations; each bar gives the range of 1 standard deviation. Symbols: ingestion of infectious chlamydiae (A); heat-inactivated chlamydiae (5); ultraviolet light-inactivated chlamydiae (0).
whereas equal multiplicities of infectious or ultraviolet-inactivated chlamydiae produced immediate damage and early death in host cells. The inability of infectious chlamydiae to promote the ingestion of heat-inactivated chlamydiae suggests that direct interaction between the parasite and the host is necessary for efficient ingestion to occur. It is tempting to speculate that this interaction involves attachment between the trypsin-sensitive components of the host and the heat-sensitive components of
I thank James W. Moulder for advice and guidance throughout this study. I am grateful to Willie Mae Conway, Karen Horoschak, and Marcia Knipher for technical assistance. I also thank Bernard Strauss for the use of his Coulter counter.
This investigation was supported by Public Health Service research grants AI-1594 and AI-13175 from the National Institute of Allergy and Infectious Diseases and Public Health Service training grant GM-603 from the National Institute of General Medical Sciences. It was also supported by an award from the Louis Block Fund of the University of Chicago. LITERATURE CITED 1. Alexander, J. J. 1968. Separation of protein synthesis in meningopneumonitis agent from that in L cells by differential susceptibility to cycloheximide. J. Bacteriol. 95:327-332. 2. Allen, J. M., and G. M. W. Cook. 1970. A study of the attachment phase of phagocytosis by murine macrophages. Exp. Cell Res. 59:105-116. 3. Armstrong, J. A., and S. E. Reed. 1967. Fine structure of lymphogranuloma venereum agent and the effect of penicillin and 5-fluorouracil. J. Gen. Microbiol. 46:435-444. 4. Cohn, Z. A., F. M. Bozeman, J. M. Campbell, J. W. Humphries, and T. K. Sawyer. 1959. Studies on growth of rickettsiae. V. Pentration of Rickettsia tsut-
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5. 6. 7. 8. 9.
10.
11. 12.
13. 14.
sugamushi into mammalian cells in vitro. J. Exp. Med. 109:271-292. David, J. R., H. S. Lawrence, and L. Thomas. 1964. The in vitro desensitization of sensitive cells by trypsin. J. Exp. Med. 120:1189-1200. Dulbecco, R., and M. Vogt. 1954. Plaque formation and the isolation of pure lines with poliomyelitis viruses. J. Exp. Med. 99:167-182. Earle, W. R. 1943. Production of malignacy in vitro. IV. The mouse fibroblast cultures and changes seen in the living cells. J. Natl. Cancer Inst. 4:165-212. 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. Grayston, J. T., and S. P. Wang. 1975. New knowledge of chlamydiae and the diseases they cause. J. Infect. Dis. 132:87-105. Hatch, T. P. 1975. Competition between Chiamydia psittaci and L cells for host isoleucine pools: a limiting factor in chlamydial multiplication. Infect. Immun. 12:211-220. Higashi, N. 1965. Electron microscopic studies on the mode of reproduction of trachoma virus and psittacosis virus in cell culture. Exp. Mol. Pathol. 4:24-39. Jones, T. C. 1975. Attachment and ingestion phases of phagocytosis, p. 269-282. In R. van Furth (ed.), Mononuclear phagocytes in immunity, infection, and pathology. Blackwell Scientific Publications, London. Kuo, C. C., and J. T. Grayston. 1976. Interaction of Chlamydia trachomatis organisms and HeLa 229 cells. Infect. Immun. 13:1103-1109. Kuo, C. C., S. P. Wang, and J. T. Grayston. 1973. Effect of polyeations, polyanions, and neuraminidase on the infectivity of trachoma-inclusion conjunctivitis and lymphogranuloma venereum organisms in iHeLa
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cells: sialic acid residues as possible receptors for trachoma-inclusion conjunctivitis. Infect. Immun. 8:74-79. 15. Ley, W. H., and V. Nussenzweig. 1968. Receptors for complement on leukocytes. J. Exp. Med. 128:9911007. 16. McLimans, W. F., E. V. Davis, F. L. Glover, and G. W. Rake. 1957. The submerged culture of mammalian cells: the spinner culture. J. Immunol. 79:428-433. 17. Manire, G. P., and J. Galasso. 1959. Persistent infection of HeLa cells with meningopneumonitis virus. J. Immunol. 83:529-533. 18. Moulder, J. W. 1969. A model for studying the biology of parasitism. Chlamydia psittaci and mouse fibroblasts (L cells). BioScience 19:875-881. 19. Moulder, J. W. 1971. The contribution of model systems to the understanding of infectious diseases. Perspect. Biol. Med. 14:486-502. 20. Moulder, J. W. 1974. Intracellular parasitism: life in an extreme environment. J. Infect. Dis. 130:300-306. 21. Moulder, J. W. 1976. Phagocytosis: the Trojan horse of intracellular parasitism. Trans. Kansas Acad. Sci., in press. 22. Moulder, J. W., T. P. Hatch, G. I. Byrne, and K. R. Kellogg. 1976. Immediate toxicity of high multiplicities of Chlamydia psittaci for mouse fibroblasts (L cells). Infect. Immun. 14:277-289. 23. Rabinovitch, M. 1967. The dissociation of the attachment and ingestion phases of phagocytosis by macrophages. Exp. Cell Res. 46:19-28. 24. Rabinovitch, M. 1969. Uptake of aldehyde-treated erythrocytes by L2 cells. Exp. Cell Res. 54:210-216. 25. Schechter, E. M. 1966. Synthesis of nucleic acid and proteins in L cells infected with the agent of meningopneumonitis. J. Bacteriol. 91:2069-2080.
AND
Vol. 14, No. 3 Printed in U.S.A.
IMMUNrrY, Sept. 1976, p. 645-651
Copyright 0 1976 American Society for Microbiology
Requirements for Ingestion of Chlamydia psittaci by Mouse Fibroblasts (L Cells) GERALD I. BYRNE
Department of Microbiology, University of Chicago, Chicago, Illinois 60637
Received for publication 23 March 1976
Ingestion of "4C-amino acid-labeled Chlamydia psittaci (6BC) by mouse fibroblasts (L cells) was inhibited when the host cells were incubated for 30 min at 37°C in Earle salts containing 10 ,ug of crystalline trypsin per ml. Tryptic digestion also inhibited the ingestion of 1-,um polystyrene latex beads. Trypsintreated L cells almost completely recovered their ability to ingest chlamydiae after 4 h at 370C in medium 199 with 5% fetal calf serum. Cycloheximide (10 ,ug/ ml) blocked this recovery. Heating 14C-amino acid-labeled C. psittaci for 3 min at 60°C inhibited its ingestion by L cells, whereas inactivating it with ultraviolet light was without effect on the ingestion rate. These results show that efficient ingestion of C. psittaci by L cells involves trypsin-labile sites on the host and heat-sensitive sites on the parasite. The failure of excess unlabeled infectious C. psittaci to promote the ingestion of "4C-labeled heat-inactivated chlamydiae suggests that direct interaction between these two sites must occur for uptake to proceed normally. The genus Chlamydia is a group of obligate intracellular procaryotes that infect a wide variety of avian and mammalian cells (18, 19). In man, they cause trachoma, inclusion conjunctivitis, lymphogranuloma venereum, psittacosis, and possibly nongonococcal urethritis (9). In both natural infections and cell cultures, chlamydiae readily gain entry into host cells that are not actively phagocytic, and the actual mechanism of their uptake has remained obscure (8, 13, 14, 21). The objective of this investigation was to learn more about the surface interactions between Chlamydia psittaci and mouse fibroblasts (L cells) that must precede ingestion of the parasite by the host cell.
ln 0.5 ln decimal fraction uninfected cells x decimal dilution = ID50 for particular number of L cells used Preparation of trypsin-treated L cells. L cells were collected from suspension by centrifugation at 500 x g for 10 min and resuspended in Earle balanced salt solution (BSS) (7) at a density of 106 cells/ ml. Measurement of the effect of treating the L cells with trypsin with respect to the rate at which they ingested C. psittaci was performed by distributing the L-cell suspensions in 5-ml volumes to 25-ml Erlenmeyer flasks that had been equilibrated in 5% CO2-95% air. Different volumes of a filter-sterilized solution of crystalline trypsin (Worthington Biochemicals) in the phosphate-buffered saline of Dulbecco and Vogt (PBS) (6) were added to duplicate flasks, and the total volume of the L-cell suspensions was adjusted to 5.5 ml with PBS. The flasks were closed with silicone rubber stoppers and shaken at 370C for 30 min at 125 strokes/min. The trypsin-treated L cells were then collected by centrifugation and resuspended to their original density in growth medium.
MATERIALS AND METHODS Growth of L cells and chlamydiae. The methods used for growing mouse fibroblasts (L cells) in suspension culture, for infecting them with C. psittaci (6BC), and for preparing harvests of C. psittaci have recently been described (10). Medium 199 supplemented with 5% heat-inactivated fetal calf serum and containing 0.1% sodium bicarbonate and 200 ,ig of streptomycin sulfate per ml was used for maintaining L cells and for propagating chlamydiae. It will be referred to simply as "medium" or "growth medium." L cells were counted in a Coulter counter, and their viability was determined by the trypan blue exclusion test (16, 17). Hatch's method (10) for titrating chlamydial infectivity was used.,It consists of determining the dose of chlamydiae needed for infecting 50% of a population of L cells of known density (IDso) according to the relation:
Preparation of "4C-labeled C. psittaci. To prepare
radioisotopically labeled chlamydial cells, the infection was carried out in medium that had been supplemented with 1 ,uCi of a "4C-L-amino acid mixture (New England Nuclear Corp.; average specific activity, 259 mCi/mmol) per ml. Incorporation of the radioisotope specifically into chlamydial protein was facilitated by addition of 2 pg of cycloheximide per ml (The Upjohn Co. ) to inhibit synthesis ofhost protein (1). The "4C-labeled C. psittaci was purified by 645
646
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INFECT. IMMUN.
enzyme digestion and differential centrifugation ac- cells infected was determined as in an ID50 titration. cording to Hatch (10). When freshly purified C. psit- Although this procedure measures uptake plus detaci was used for infection at a multiplicity of 1 to 10 velopment to a microscopically visible inclusion, it ID50 per host cell, more than 80% of the precipitable gives a reasonable reflection of the rate of chlam14C label became associated with L cells after 2 h at ydial ingestion when 1 ID50 per cell is used. Only 370C. This represented essentially all of the infec- 50% of the L cells are infected at a maximum, and tious chlamydiae present as determined by retitra- most of the host cells contain only a single inclusion. tion of the supernatant fluid after a single centrifu- Thus, multiply infected cells are not a complicating gation at 500 x g for 10 min. Labled C. psittaci factor, and one infected cell comes close to reprepreparations equivalent to 5 x 106 L-cell ID50 doses senting one ingestion event. Quantitating ingestion had counts in the range of 400 to 800 cpm. by this method also obviates the problem of distinInactivation of C. psittaci with heat and ultravi- guishing between external attachment and true upolet light. Chlamydiae that had been grown in unla- take, since only those chlamydiae that are actually beled or in '4C--amino acid-labeled medium were taken into the L cells are able to replicate and form harvested from infected L cells, purified as just de- inclusions. Infected cell counts were made by somescribed, and suspended in PBS. Two milliliters of one who was not aware of the origin of the samples the suspension was then placed in a plastic petri counted. dish with a surface area of 28 cm2 and inactivated Measurement of the ingestion of polystyrene lawith ultraviolet light (3-min exposure to a 30-W tex spheres. When the ingestion of polystyrene latex General Electric germicidal lamp at'a distance of 76 spheres (Dow Diagnostics; 1-,um diameter) was to be cm). A 2-ml portion of the PBS suspension was measured, L cells were suspended in growth meplaced in a 13-mm-diameter tube and shaken for 3 dium to a density of 106 cells/ml and distributed into min in a 60°C water bath and then cooled in an ice flasks as already described. Latex spheres were then bath. Such heating did not macroscopically or mi- added in a ratio of 500 spheres for every L cell. croscopically clump the chlamydial cells and did not Samples were shaken at 370C for 60 min and washed alter their sedimentation properties. three times with growth medium to remove unassoMeasurement of the ingestion of 14C-labeled chla- ciated spheres from the cells. The L cells were then mydiae. For experiments in which the ingestion of diluted to a density of 0.25 x 106 cells/ml in growth 14C-labeled chlamydiae was to be measured, appro- medium containing 10% heat-inactivated fetal calf priately treated L cells were suspended to a density serum and plated out in plastic tissue culture flasks of 106 cells/ml in growth medium. The cell suspen- at a density of 5 x 104 cells/cm2. After 4 h at 370C, sion was then distributed in 5-ml portions to equili- the monolayers were washed three times with PBS brated 25-ml flasks, and 1 ID50 per L cell of a freshly and fixed in absolute ethanol. The number of titered 14C-labeled C. psittaci suspension was added spheres ingested by 200 L cells was then counted by to each flask. The flasks were shaken at 370C for 30 phase-contrast microscopy. Sphere counts were to 120 min. The L cell-chlamydiae mixtures were made by someone who did not known the nature of then centrifuged at 4°C for 5 min at 300 x g. This the samples counted. provided sufficient. force to sediment the L cells and Demonstration of the regeneration of trypsinL-cell-associated chlamydiae, but not enough force labile sites on L cells. These experiments were done to sediment free, unattached chlamydiae. To be sure by treating L cells with 10 ,ug of trypsin per ml as that all the unassociated chlamydiae had been described in a earlier section. The cells were centriwashed from the host cells, the L-cell pellet was fuged, resuspended to a density of 106 cells per ml of resuspended in ice-cold growth medium and recen- growth medium, and transferred to Erlenmeyer trifuged. This procedure was repeated an additional flasks in 5-ml volumes. Another part of the same Ltime. Monitoring the radioactivity in the successive cell population was carried through the same masupernatant fluids showed that all the free chlamy- nipulations without the addition of trypsin, and the diae were removed by three washes. After the third same number of flasks was prepared for these uncentrifugation, the L cells were resuspended in 2 ml treated L cells. Four untreated and four trypsinof 5% trichloroacetic acid, and the precipitable treated flasks were set up for each recovery period to counts were collected on membrane filters (Milli- be tested. Cycloheximide at a final concentration of pore Corp.) by a previously published procedure (1). 10 Ag/ml was added to two of each set of four flasks. Samples were counted in 5 ml of a toluene-based The L-cell suspensions were shaken at 370C; after 0, scintillant in a Packard Tri-Carb liquid scintillation 30, 60, 120, and 240 min, 1 ID50 per L cell of either unlabeled or 14C-labeled C. psittaci was added, and spectrometer. Measurement of the ingestion of unlabeled shaking was continued for another 60 min. At apchlamydiae. Infection with 1 IDO per host cell, propriate times, samples were removed from the incubation, and washing the infected L cells by cen- shaker, and the uptake of labeled or unlabeled C. trifugation were carried out as just described. The psittaci was determined as described earlier. final L-cell pellet was resuspended to original denEffect of excess unlabeled, infectious C. psittaci sity in growth medium containing 10% heat-inacti- on the ingestion of '4C-labeled, heat-killed chlamyvated fetal calf serum and was diluted fourfold in diae. In these experiments, 5 or 50 ID50 per L cell of the same medium, and 1-ml volumes were added to unlabeled, purified, infectious chlamydiae was duplicate cover slip-containing Leighton tubes (25), mixed with 1 ID50 of 14C-labeled, purified, heat-inacwhich were incubated for 18 to 24 h at 370C in 5% tivated C. psittaci, and samples were added to dupliC02-95% air. They were then stained with Giemsa cate flasks containing 5 ml of an L-cell suspension at stain according to Hatch (10). The percentage of L 106 cells/ml. Ingestion of the 14C-labeled C. psittaci
VOL. 14, 1976
CHLAMYDIAL INGESTION BY L CELLS
was followed by the procedure already described. Samples that contained 1 ID,0 of infectious, 14Clabeled chlamydiae and samples that contained the 14C-labeled, heat-killed chlamydiae alone were included in each experiment.
RESULTS Inhibition of the ingestion of C. psittaci by tryptic digestion of L cells. When L cells were suspended in Earle balanced salt solution, treated with trypsin for 30 min at 370C, and resuspended in growth medium, as little as 1 ,ug of trypsin per ml inhibited the ingestion of infectious C. psittaci (Fig. 1). A maximum inhibition of more than 80% was obtained with 10 ,ug of trypsin per ml. When the L cells were treated with trypsin while suspended in growth medium containing 5% heat-inactivated fetal calf serum, a potent inhibitor of tryptic activity, no decrease in the ability to ingest chlamydiae was observed. Recovery of the ability to ingest C. psittaci and its inhibition by cycloheximide. When the
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FIG. 2. Recovery of the ability of trypsin-treated L cells to ingest C. psittaci and inhibition of recovery by cycloheximide: use of unlabeled chlamydiae to measure ingestion. Ingestion of unlabeled chlamydiae was followed by the procedure given in Materials and Methods. One hundred percent uptake was the ingestion ofunlabeled infectious C. psittaci found at 1 h in populations ofcells that were not treated with trypsin or with cycloheximide and were not preincubated in recovery medium. Each point is the mean ofsix determinations, and each bar represents ± 1 standard deviation. Symbols: no trypsin treatment and no cycloheximide in recovery medium (O); no trypsin treatment and 10 pg ofcycloheximide per ml in recovery medium (U); 10 pg of trypsin per ml for 30 min at 370C and no cycloheximide in recovery medium (0); trypsin treatment and cycloheximide in recovery medium (O).
observed that Rickettsia tsutsugamushi easily entered mouse lymphoblasts and fibroblasts (L cells) but that, under similar conditions, Escherichia coli and Staphylococcus aureus got into these host cells only with great difficulty. L cells also ingest C. psittaci much more efficiently than they do most other microorganisms. In experiments not described here, E. coli was labeled with "4C-amino acids to such an extent that ingestion of less than 1 bacterial cell per L cell could have been detected. Yet when L cells were exposed to labeled E. coli in a ratio of 1,000 bacterial cells per L cell, the L cell-associated label after 2 h at 370C was not significantly greater than that of the 0-h control. In contrast, when "4C-labeled C. psittaci were added to L-cell suspensions under identical conditions, nearly half of the chlamydiae were ingested when 1,000 C. psittaci were added per L cell, and essentially all of the chlamydiae were ingested when the ratio of chlamydial cells to L cells was less than 100:1. Therefore the principal objective of this investigation was to find out why C. psittaci, like many other obligate intracellular parasites, is ingested with unusual efficiency by cells that do not readily phagocytose most other microorganisms. There is little doubt that C. psittaci enters L cells by phagocytosis. Inside the host cell, the chlamydiae always lie within membrane-bound vacuoles assumed to be phagosomes (3, 8, 11), and the metabolic energy for chlamydial entry is derived from the L-cell host and not from the parasite (8, 22). The disproportionate efficiency of chlamydial ingestion by L cells may be considered an example of induced phagocytosis, a term proposed by Jones (12) to describe the entry of Toxoplasma gondii into nonprofessional phagocytes. Precise mechanisms for induced phagocytosis have not been described. The profound interference with phagocytosis of C. psittaci by L cells brought about by treating the host cells with trypsin before exposing TABLE 1. Inhibition of the ingestion ofpolystyrene
latex spheres by trypic treatment of L cellsa obligate intracellular parasites is the ability to promote their ingestion by host cells that are No. of spheres Treatment of L cells not actively phagocytic (21). Jones (12) and Racell ingested/L binovitch (24) have called these cells facultative None ..... ............. 15.3 or nonprofessional phagocytes. This property Cycloheximide, 10 lug/ml ............... 14.2 enables an obligate intracellular parasite to Trypsin, 10 ,ug/ml .................. 2.8 occupy an unexploited ecological niche (20) and Cycloheximide and trypsin ............. 2.6 to avoid the potent constitutive and inducible a L cells were treated with trypsin as described in intracellular defense mechanisms of the profes- Materials and Methods. The procedure for measursional phagocytes. Obligate intracellular pro- ing ingestion of 1-,um-diameter polystyrene latex caryotes also enter established lines of mamma- spheres was also described in Materials and Methlian cells with much greater facility than do ods. Each value represents the mean of duplicate ordinary bacteria. For example, Cohn et al. (4) determinations.
CHLAMYDIAL INGESTION BY L CELLS
VOL. 14, 1976 A
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them to the chlamydial cells shows that there are trypsin-sensitive structures on the L-cell surface essential for chlamydial uptake. Rabinovitch (23) has separated phagocytosis by macrophages into phases of attachment and ingestion. However, Friis (8) was unable to demonstrate attachment of C. psittaci to L cells in the absence of ingestion, either by using 14C-labeled chlamydiae or thin-section electron microscopy. It will be assumed that trypsin treatment interfered with an attachment of C. psittaci to L cells that is immediately followed by ingestion. This is supported by the agreement between the two methods used to assay the trypsin effect, one of which depended on attachment only (changes in L-cell-associated, 14Clabeled C. psittaci) and the other on both attachment and ingestion (changes in the percentage of L cells infected at the IDo end point). The structures destroyed by trypsin are probably recognition sites roughly analogous to the trypsin-sensitive sites on macrophages that recognize migration inhibition factor (5), complement (15), and Bacillus subtilis (2). The destruction of these sites by trypsin and the inhibition of their regeneration by cycloheximide suggests that they are protein or glycoprotein in nature. These experiments do not permit further conclusions as to the nature of the trypsin-sensitive recognition sites. The inhibition of
the ingestion of both chlamydial cells and polystyrene latex spheres by trypsin suggests that they are nonspecific. The marked reduction in the rate of phagocytosis of C. psittaci that had been heated at 60°C for 3 min is best explained by assuming that denaturation of heat-labile areas on the chlamydial cell surface destroyed its ability to induce rapid phagocytosis by L cells. The failure of inactivation with ultraviolet light to produce a comparable drop in the rate of chlamydial ingestion shows that loss of infectivity cannot in itself explain the impaired phagocytosis of heated C. psittaci. The inactivation of these putative attachment sites by brief heating at relatively low temperature suggests that they, like the recognition sites on L cells, are protein in nature. Friis (8) observed some reduction in the rate of ingestion of heated C. psittaci by L cells, but he concluded that it was not significant. However, in this investigation, repeated comparisons of the rate of phagocytosis of infectious and heat-inactivated C. psittaci showed that the heated chlamydial cells were ingested at significantly slower rates. Other experiments also demonstrated an altered reactivity of heated C. psittaci toward L cells. As described in another paper (22), very high multiplicities of heated C. psittaci had no harmful effect on L cells,
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the parasite, but further work will be necessary before any substantial conclusions can be drawn. Cohn et al. (4) studied phagocytosis of R. tsutsugamushi by mouse lymphoblasts and fibroblasts. Ingestion of rickettsiae was inhibited by inactivation either by heat or by ultraviolet light. In addition, compounds related to glutamate, which is probably oxidized by these organisms, enhanced ingestion, whereas inhibitors of glutamate oxidation depressed it. Kuo et al. (14) studied the entry of trachoma and lymphogranuloma venereum strains of Chlamydia trachomatis into HeLa 229 cells. Ingestion of trachoma strains was enhanced by pretreatment of host cells with polycations; the entry of lymphogranuloma venereum strains was inhibited. Treatment of the HeLa cells with neuraminidase reduced the uptake of trachoma strains and was without effect on lymphogranuloma venereum strains. An excess of heated trachoma cells (but not lymphogranuloma venereum cells) blocked the ingestion of infectious trachoma agent. Kuo and Grayston (13) found that heat inactivation of both trachoma and lymphogranuloma venereum strains of C. trachomatis inhibited their ingestion by HeLa 229 cells. Although it appears unlikely that a single mechanism of induced phagocytosis can explain all these diverse observations, the generalization that a heat-labile parasite component is essential for the efficient ingestion of all kinds of chlamydiae now seems to be justified. ACKNOWLEDGMENTS
MINUTES AFTER INFECTION
FIG. 4. Ingestion of infectious, heat-inactivated, and ultraviolet-inactivated 14C-amino acid-labeled C. psittaci by L cells. Methods for inactivating C. psittaci and for following the ingestion of labeled chlamydial cells are described in Materials and Methods. One hundred percent uptake was the ingestion of 14C-labeled infectious C. psittaci obtained at 3 h of incubation. Each point represents the mean of six determinations; each bar gives the range of 1 standard deviation. Symbols: ingestion of infectious chlamydiae (A); heat-inactivated chlamydiae (5); ultraviolet light-inactivated chlamydiae (0).
whereas equal multiplicities of infectious or ultraviolet-inactivated chlamydiae produced immediate damage and early death in host cells. The inability of infectious chlamydiae to promote the ingestion of heat-inactivated chlamydiae suggests that direct interaction between the parasite and the host is necessary for efficient ingestion to occur. It is tempting to speculate that this interaction involves attachment between the trypsin-sensitive components of the host and the heat-sensitive components of
I thank James W. Moulder for advice and guidance throughout this study. I am grateful to Willie Mae Conway, Karen Horoschak, and Marcia Knipher for technical assistance. I also thank Bernard Strauss for the use of his Coulter counter.
This investigation was supported by Public Health Service research grants AI-1594 and AI-13175 from the National Institute of Allergy and Infectious Diseases and Public Health Service training grant GM-603 from the National Institute of General Medical Sciences. It was also supported by an award from the Louis Block Fund of the University of Chicago. LITERATURE CITED 1. Alexander, J. J. 1968. Separation of protein synthesis in meningopneumonitis agent from that in L cells by differential susceptibility to cycloheximide. J. Bacteriol. 95:327-332. 2. Allen, J. M., and G. M. W. Cook. 1970. A study of the attachment phase of phagocytosis by murine macrophages. Exp. Cell Res. 59:105-116. 3. Armstrong, J. A., and S. E. Reed. 1967. Fine structure of lymphogranuloma venereum agent and the effect of penicillin and 5-fluorouracil. J. Gen. Microbiol. 46:435-444. 4. Cohn, Z. A., F. M. Bozeman, J. M. Campbell, J. W. Humphries, and T. K. Sawyer. 1959. Studies on growth of rickettsiae. V. Pentration of Rickettsia tsut-
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10.
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13. 14.
sugamushi into mammalian cells in vitro. J. Exp. Med. 109:271-292. David, J. R., H. S. Lawrence, and L. Thomas. 1964. The in vitro desensitization of sensitive cells by trypsin. J. Exp. Med. 120:1189-1200. Dulbecco, R., and M. Vogt. 1954. Plaque formation and the isolation of pure lines with poliomyelitis viruses. J. Exp. Med. 99:167-182. Earle, W. R. 1943. Production of malignacy in vitro. IV. The mouse fibroblast cultures and changes seen in the living cells. J. Natl. Cancer Inst. 4:165-212. 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. Grayston, J. T., and S. P. Wang. 1975. New knowledge of chlamydiae and the diseases they cause. J. Infect. Dis. 132:87-105. Hatch, T. P. 1975. Competition between Chiamydia psittaci and L cells for host isoleucine pools: a limiting factor in chlamydial multiplication. Infect. Immun. 12:211-220. Higashi, N. 1965. Electron microscopic studies on the mode of reproduction of trachoma virus and psittacosis virus in cell culture. Exp. Mol. Pathol. 4:24-39. Jones, T. C. 1975. Attachment and ingestion phases of phagocytosis, p. 269-282. In R. van Furth (ed.), Mononuclear phagocytes in immunity, infection, and pathology. Blackwell Scientific Publications, London. Kuo, C. C., and J. T. Grayston. 1976. Interaction of Chlamydia trachomatis organisms and HeLa 229 cells. Infect. Immun. 13:1103-1109. Kuo, C. C., S. P. Wang, and J. T. Grayston. 1973. Effect of polyeations, polyanions, and neuraminidase on the infectivity of trachoma-inclusion conjunctivitis and lymphogranuloma venereum organisms in iHeLa
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cells: sialic acid residues as possible receptors for trachoma-inclusion conjunctivitis. Infect. Immun. 8:74-79. 15. Ley, W. H., and V. Nussenzweig. 1968. Receptors for complement on leukocytes. J. Exp. Med. 128:9911007. 16. McLimans, W. F., E. V. Davis, F. L. Glover, and G. W. Rake. 1957. The submerged culture of mammalian cells: the spinner culture. J. Immunol. 79:428-433. 17. Manire, G. P., and J. Galasso. 1959. Persistent infection of HeLa cells with meningopneumonitis virus. J. Immunol. 83:529-533. 18. Moulder, J. W. 1969. A model for studying the biology of parasitism. Chlamydia psittaci and mouse fibroblasts (L cells). BioScience 19:875-881. 19. Moulder, J. W. 1971. The contribution of model systems to the understanding of infectious diseases. Perspect. Biol. Med. 14:486-502. 20. Moulder, J. W. 1974. Intracellular parasitism: life in an extreme environment. J. Infect. Dis. 130:300-306. 21. Moulder, J. W. 1976. Phagocytosis: the Trojan horse of intracellular parasitism. Trans. Kansas Acad. Sci., in press. 22. Moulder, J. W., T. P. Hatch, G. I. Byrne, and K. R. Kellogg. 1976. Immediate toxicity of high multiplicities of Chlamydia psittaci for mouse fibroblasts (L cells). Infect. Immun. 14:277-289. 23. Rabinovitch, M. 1967. The dissociation of the attachment and ingestion phases of phagocytosis by macrophages. Exp. Cell Res. 46:19-28. 24. Rabinovitch, M. 1969. Uptake of aldehyde-treated erythrocytes by L2 cells. Exp. Cell Res. 54:210-216. 25. Schechter, E. M. 1966. Synthesis of nucleic acid and proteins in L cells infected with the agent of meningopneumonitis. J. Bacteriol. 91:2069-2080.
