Electron microscopy of the in vivo internalization of virulent Chlamydia psittaci 6BC strain1
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NONNAKORDOVP;,JOHNC. WILT, LYNN BURTON,A N D CAROLINE MARTIN Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba Accepted February 21, 1975
KORDOVL, N., J. C. WILT,L. BURTON,and C. MARTIN.1975. Electron microscopy of the in vivo internalization of virulent Chlamydia psittaci 6BC strain. Can. J . Microbiol. 21: 945-953. The internalization of virulent Chlamydia psittaci 6BC particles by wandering mononuclear phagocytes in the peritoneal cavity of intraperitoneally inoculated mice occurred asynchronously, i.e., fragile reticulate bodies (RB) appeared to be more readily phagocytized than the rigid elementary bodies (EB). Early damage of mononuclear phagocytes occurred after internalization of chlamydiae. This was followed by a decreased uptake of particles, and may explain the relatively long persistence (up to 6 h after inoculation) of free, extracellular, "swollen," and RB-like particles. Internalized particles within phagolysosomes showed varying degrees of disintegration. The subsequent influx of polymorphonuclear phagocytes and monocytes into the inflammed peritoneal cavity may explain the rapid disappearance of chlamydiae and their antigens from the peritoneal fluid. The alteration in ultrastructure of peritoneal cells and chlamydial parasites during the inflammatory process are discussed. KORDOVL, N., J. C. WILT,L. BURTONet C. MARTIN.1975. Electron microscopy of the in vivo internalization of virulent Chlamydia psittaci 6BC strain. Can. J. Microbiol. 21: 945-953. Chez des souris inoculees par voie intrapkritoneale le passage des particules virulentes de Cl~lamydiapsittaci 6BC B I'interieur des mononucleaires circulants dans la cavitC peritontale se fait de fagon asynchrone. Les corps rtticulks fragiles (RB) semblent en effet phagocytes plus rapidement que les corps eltmentaires rigides (EB). Aprks la penetration des chlamydies, les phagocytes mononuclCaires sont rapidement endommagks. Cela entraine une diminution de la captation des particules et peut expliquer la persistance relativement longue (jusqu'a 6 h aprks I'inoculation) de particules extracellulaires "gonflCes" et d'allure RB. Les particules intracellulaires incluses dans des phagolysosomes sont dksintegrees a differents degrCs. L'arrivee subskquente des polynucleaires et des monocytes dans la cavitt peritoneale inflammee peut expliquer la disparition rapide dans le liquide peritoneal des chlamydies et de leurs antigknes. On discute de I'altCration de I'ultrastructure des cellules pCritonCales et des parasites chlamydiaux au cours du processus inflammatoire. [Traduit par le journal]
Introduction contrast, after infection of mice with an aviruIn our previous in vivo studies of chlamydiae lent 6BC strain readily identifiable parasites the response of wandering mouse peritoneal were observed in mononuclear phagocytes. The phagocytes and the fate of either a virulent or lysosomes of macrophages were refractory to avirulent (for mouse) Chlamydia psittaci 6BC avirulent 6BC particles that were initially instrain has been followed with cytochemical gested and some macrophages were "transmethods by light microscopy (22, 23) and im- formed" into large epithelioid cells containing munofluorescence (21, 24). We described the chlamydial inclusions in the cytoplasm (23). The objective of the present study was to virulent 6BC strain (grown in L cells) as inducing observe the relationship between parasites and an early injurious effect on mononuclear phagol wandering phagocytes at the ultrastructural cytes and their lysosomes; a marked influx of polymorphonuclear phagocytes (PMN's) oc- level during the early crucial phases after intracurred which showed early karyorrhexis and peritoneal inoculation of mice with virulent 6BC lysis. GimCnez or Giemsa staining failed to detect chlamydiae in peritoneal fluids up to 6 Material and Methods days after inoculation of mice. Chlamydiae The C . psittaci 6BC strain (grown in L cells), the CF-1 were recoverable from the peritoneal fluid only strain of mice, and the procedures of inoculation and during the 1st h after inoculation of mice. In harvesting peritoneal cells used throughout these studies 'Received August 29, 1974.
were the same a s described in the preceding paper (22). For ultrastructural studies, samples of semipurified
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chlamydiae and peritoneal cells from infected and control mice were processed by methods described by others (16) and used in our previous studies (20, 33). Mice were injected intraperitoneally with 0,2 ml of semipurified chlamydial inoculum as described before (22) and killed at medetermined time intervals after inoculation. Three miililitres of fresh 1% glutaraldehyde (Ladd Research Industries) in phosphate buffer at a pH of 7.0 was injected into the peritoneal cavity, the fluid was withdrawn and centrifuged at 1000 rpm for 20 min, and the pellet was processed for electron microscopy. Thin sections were obtained using a LKB ultramicrotome with a glass knife, picked up on 400-mesh copper specimen grids, and double-stained with uranyl acetate and lead citrate. A Philips 201 electron microscope was used to examine the material. Micrographs were made at magnifications up to 8640 diameters and then enlarged photographically. Other methods are described along with Results.
Results Differential counts of peritoneal cells after inoculation of mice with virulent 6BC chlamydiae have shown that few PMN's were present in the peritoneal cavity up to 6 h after inoculation. The early events of the interaction are therefore predominantly concerned with mononuclear phagocytes. The polymorphic chlamydial particles (5) present in thin sections (Figs. 9 and 10) were differentiated in accordance with others (5,25,26) into three main forms: small (0.25-0.35 microns (p) in diameter), dense, centered, rigid, elementary bodies (EB), large, (0.5 p) nonrigid, reticulate bodies (RB), and intermediate (0.35-5 p) bodies (IB). The particles were identified in their relation to phagocytes as completely unattached, as present in invaginations, as attached, or as intracellular. As early as 5 min after intraperitoneal inoculation of chlamydiae, RB and IB were seen within phagocytes surrounded by an intactappearing cellular membrane (Fig. 1) most probably derived from the surface membrane of the phagocyte. Several EB appeared to be attached to phagocytes (Fig. 1). At 5 min most of the organisms were unattached (Fig. 11). This is in agreement with assays of chlamydial infec-
tivity performed in parallel in which high titers of infective parasites were found in the fractions of peritoneal fluids collected 5 rnin after inoculation (22). At 10 rnin after inoculation there was a marked increase in the number of EB and IB attached to cells (Fig. 3). Internalized EB and IB showing varying degrees of ultrastructural alterations were localized in different compartments in the cytoplasm of the phagocytes with an average of three to eight particles per phagocyte profile (Figs. 4 and 8). Large, well-defined, internalized RB were rarely detected at 10 rnin or later after inoculation although they were seen extracellularly (Figs. 12 and 13). In some cells particles appeared to be closely surrounded by a cellular membrane (Fig. 2), in some the particles were present singly or in clusters in large vacuoles, and in other cells the particles were detected in dense cytoplasmic areas with no sharp boundary between them and the surrounding cytoplasm (Fig. 8). Most phagocytes with or without chlamydiae exhibited alterations of the lysosomal apparatus (10, 11) including incomplete or fragmentary vesicular membranes; some vesicles were empty and others contained granular material or dense bodies (Fig. 8). Despite the increased uptake of particles, collections of unattached chlamydial particles remained in the fluid interspersed amongst dense granular material, small empty vesicles, residual bodies, fragments of membranes, and lamellar structures (Figs. 5 and 6). Extracellular particles harvested from peritoneal fluids at predetermined times after inoculation appeared agglutinated (Fig. 12) although they were dispersed in the inoculum before inoculation. In contrast to the relatively large number of EB inside phagocytes at 10 rnin after inoculation, few EB could be detected in phagocytes 30 rnin after inoculation. Particles were often located in large peripheral vacuoles, most probably invaginations of the outer cytoplasmic membrane (Fig. 5). Many unattached EB were also
ABBREVIATIONS USED: MP, mononuclear phagocyte; M, mitochondrion; N, nucleus; L, lysosome. The bar on all micrographs represents 0.1 p. FIG. 1. Internalized RB in MP 5 min after inoculation of mice. Fig. 2. Internalized IB and EB 10 min after inoculation of mice. FIG. 3. EB appear attached to the outer membrane of M P 10 min after inoculation. FIG. 4. EB (arrows) are entrapped between microvilli of MP. There is some variation in the density of the cytoplasm of EB enclosed by two closely apposed double-track structures. FIGS.5-6. Localization of EB and different ultrastructural alterations of EB and MP 30 rnin after inoculation. FIG. 7. Disintegration of EB (arrows) in phagolysosomes of M P 6 h after inoculation.
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KORDOVA ET AL.: CHLAMYDIA PSITTACI 6BC STRAIN
CAN. J. MICROBIOL. VOL. 21, 1975
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FIG.8. Fate of EB (arrows) in different compartments of MP 10 min after inoculation.
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KORDOVA ET AL.: CHLAMYDIA PSITTACI 6BC STRAIN
FIGS.9-10, EB and RB in semipurified suspension before inoculation. FIGS.11-14. Alterations in the ultrastructure of extracellular chlamydia1 particles in peritoneal fluid; FIGS.1 1 and 12, 5 min after inoculation; FIG. 13, 10 min after inoculation: and FIG. 14, 1 h after inoculation.
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seen interspersed between phagocytes (Fig. 6). Most phagocytes showed ultrastructural alterations such as large perinuclear and peripheral vacuolization, paucity or alteration of cytoplasmic organelles, dense and granular cytoplasmic areas, thinning of ground cytoplasm, and complete cellular disintegration. Peritoneal fluid collected at 1 and 6 h after inoculation required extensive screening to detect a few severely damaged intracellular particles (Fig. 7). It is noteworthy that extracellular accumulations of agglutinated, severely altered particles sometimes entrapped in whorls of membranes were detected on several occasions. The ultrastructural alteration of particles undergoing disintegration in phagolysosomes is seen in Figs. 7 and 8. Many cell-free particles recovered from peritoneal fluids at 1 and 6 h appeared "swollen" or "fuzzy" and RB-like (Fig. 14); their surface envelopes appeared loosened or to be undergoing dissolution. In some particles retraction and in others rarefication of the cytoplasm was seen. No chlamydiae or a few chlamydiae-like structures were seen in peritoneal fluids collected at day 1 and up to 6 days after inoculation, i.e. at a time when the marked influx of PMN's occurred in the inflammed peritoneum (22). In contrast to electron microscopic observations, the greatest amount of cell-bound chlamydial infectivity was detected at 1 h after inoculation. No infectivity was identified in the cell-free fraction of peritoneal fluids at this time. No infectivity was identified in either cell-bound or cell-free fractions sampled at 6 h, 1 day, and daily up to 6 days after inoculation. The observations as described above have shown that the internalization of virulent 6BC chlamydial particles by wandering mononuclear phagocytes in the peritoneal cavity of intraperitoneally inoculated mice occurred asynchronously. Fragile RB and IB appeared to be more readily phagocytized than the rigid EB. After internalization of chlamydiae early damage of mononuclear phagocytes occurred. This apparently caused a decreased uptake of particles and may explain the relatively long persistence (up to 6 h after inoculation) of unattached extracellular particles. Internalized particles were disintegrated in phagolysosomes. The increased influx of PMN's and monocytes from the circulation into the inflammed peritoneal cavity might
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explain the subsequent rapid disappearance of chlamydial particles and their antigens (22, 24) from the peritoneal fluid examined later, up to 6 days after inoculation.
Discussion The early chlamydiae-phagocyte interaction in vivo has not been analyzed by electron microscopy before the present study. Some in vivo ultrastructural studies during the later stages of infection have been described in connection with intestinal chlamydial infections of calves (12, 13, 14) and intraarticular infection of lambs (9) in which the authors observed a varying response of macrophages to chlamydiae. Our studies have demonstrated that the strain of chlamydiae and its virulence for the mouse strongly affect the outcome of the chlamydiae-phagocyte relationship (22, 23). After inoculation of virulent 6BC chlamydiae an early ultrastructural alteration developed in both mononuclear phagocytes and in the parasites, with subsequent rapid loss of the chlamydial infectivity. In an earlier study Drobyshevskaya and co-workers (15) demonstrated a rapid inactivation of chlamydial infectivity in the peritoneal cavity of mice using two virulent chlamydial strains, but electron microscopy was not performed. Electron microscopy has revealed several patterns of response of phagocytes to microbes depending on the virulence of the parasite and on the degree of resistance of the phagocyte (1,2, 3, 32). A frequent response to the uptake of microbes is the formation of phagolysosomes with the subsequent digestion of microbes. This pattern has been described in mouse peritoneal macrophages and in human macrophages in several bacterial infections (1, 32). Less frequently virulent, toxic microbes affect the integrity of the phagocyte membrane (1, 2, 32). It is not clear how lysosomes injure the cell of which they constitute a part or how they affect surrounding tissues (I I). It appears that under some circumstances direct injury to lysosomes induces damage to cellular structures leading to eventual cell death. Release of enzymes from disrupted lysosomes through the cell membrane by a kind of exocytosis has been suggested as one of several possible mechanisms for damage of surrounding structures. Evidence has also been obtained for the presence of several tissue-
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damaging substances especially in leucocyte lysosomes (1 1, 38, 39). Because of the agglutinogens of chlamydial EB (6, 35) one might suggest that their injurious action upon phagocytes may be initiated on the surface of certain phagocytes. If this indeed is the case, the possibility exists that at least in some phagocytes, EB, instead of being engulfed, may be eluted because of the inhibited phagocytic activity of such injured phagocytes. Furthermore, injury of the cell surface per se may elicit release of lysosomal enzymes inside and outside the cell (2, 11). One can therefore not dismiss the possibility that the structure and function of the chlamydial EB may be altered during adsorption to some phagocytes and before subsequent uptake by other phagocytes. It is possible to speculate that the accumulation of cell-free ultrastructurally damaged RB-like chlamydial particles in the peritoneal fluid could be attributed to the early damage of the surface of the phagocyte membrane and release of lysosoma1 enzymes as well as tissue-damaging substances from these cells. Lysozyme is usually associated with the lysosomal granule of host cells, particularly in phagocytes (8). Lysosomal extracts and lysosomal enzymes such as lysozyme, leucozyme, acid phosphatase, and phospholipase have produced fragile forms from gram-positive as well gram-negative bacteria (4, 29). Macrophages have been reported to exert a lysozyme-like activity on Brucella (29); lysosomal extracts from PMN's converted smooth Brucella to spheroplasts and killed artificially induced spheroplasts in vitro. Egg white lysozyme has not elicited this reaction, thus indicating the presence of anti-Brucella substances in phagocyte lysosomes (29). In this connection it is noteworthy that a limited degree of chlamydial infectivity has been reported in chlamydiaeinfected tissue cultures after treatment with chicken egg white lysozyme (19). It is generally accepted as an oversimplification that chlamydial populations consist of two kinds of particles; the infectious, rigid EB which reorganizes after phagocytosis and during the "development cycle" into noninfectious, fragile RB. The mechanism whereby the small infectious EB transforms intracellularly into the large noninfectious cell wall deficient form and back to the small form again is largely unknown. In the present studies fragile RB and (or) IB
PSITTACI 6BC STRAIN
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appeared to be more readily phagocytized by mononuclear phagocytes than the rigid EB. This raises several questions. Whatever the nature of RB, evidence is accumulating (7, 30, 34, 36, 37) that the surface envelopes of fragile RB differ physically and chemically from that of the rigid EB. It has been shown recently (35) that hemagglutinogen (6) present in high titers in homogenates of EB and their cell walls was almost absent in preparation of purified RB. The fragility of RB has been attributed to a lack of the rigidity factor in the surface envelopes of these large particles (37). Whatever the basis of the cell wall deficiency of RB may be, it is now well established that in general the behavior of cell wall deficient microbes in vivo is different from that of their original rigid classical forms. Some cell wall deficient variants are for example "indifferent" to certain antimicrobial and host tissue factors (28). Furthermore, some cell wall deficient bacterial variants are rather rapidly phagocytized (17) and also survive within macrophages and monocytes (1 8,32). It has been demonstrated (32) that simultaneous exposure of macrophages to more than one kind of particle can influence phagocytosis and also clearance by these cells. If indeed the fragile RB are more rapidly phagocytized than the rigid EB in certain cells, the possibility exists that in a mixed population of chlamydial strains the RB ingested initially could interfere with the subsequent uptake of EB. Electron microscopic studies have demonstrated that the proportion of RB in the overall population of chlamydiae varies in different hosts (27, 31) but the reasons for these population changes are unknown.
Acknowledgments This research was supported by a Medical Research Council grant No. MA3901. Appreciation is expressed to Mrs. Eleanor Shewchuk and Mrs. Esther Elias for assistance. 1. ALLEN,J . M. 1969. Lysosomes in bacterial infection. In Lysosomes in biology and pathology. Frontiers of biology series: 14B. Edited b y J. T . Dingle and H. B. Fell. North-Holland Publishing Company, Amsterdam and London. pp. 41-68. A. 1967. Lysosomes in virus-infected cells. 2. ALLISON, In Perspectives in virology. V. Virus directed host response. Edited b y V. POLLARD.Academic Press, Inc., New York and London. pp. 29-60. 3. ALLISON, A. C., and L. MALLUCCI. 1%5. Histochemical studies of lysosomes and lysosomal enzymes in
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virus-infected cell cultures. J. Exp. Med. 121: 463-474. AMANO,T . , Y. S E K I ,K. FUJIKAWA, S. KASHIBA, T. MORIOKA, and S. ICHIKAWA. 1956. The isolation and characterization of protoplasts from Escherichia coli B with the treatment of leucocytes extract. Med. J. Osaka Univ. 7: 245-256. ANDERSON, D. R., H. E. HOPPS,M. F. BARILE, and B. C. BERNHEIM. 1965. Comparison of the ultrastructure of several rickettsiae, ornithosis virus, and mycoplasma in tissue culture. J. Bacteriol. 90: 1387-1404. BARRON, A. L., and M. C. RIERA.1%9. Studies on hemagglutination by Chlamydia. Proc. Soc. Exp. Biol. Med. 131: 1087-1090. G., and G. P. MANIRE.1%9. The CHRISTOFFERSEN, toxicity of meningopneumonitis organisms (Chlamydia psittaci) at different stages of development. J. Immunol. 103: 1085-1088. COHN,Z. A,, J . G. HIRSCH,and E. WIENER.1%3. Lysosomes and endocytosis. The cytoplasmic granules of phagocytic cells and the degradation of bacteria. I n Ciba foundation symposium on lysosomes. Edited by A. V. S. Rench and M. P. Cameron. J . and A. Churchill, Ltd., London. p. 126. CUTLIP,R. C. 1974. Ultrastructure of the synovial membrane of lambs affected with chlamydial polyarthritis. Am. J . Vet. Res. 35: 171-176. 1%9. DAEMS,W. TH., E. WISSE,and P. BREDEROO. Electron microscopy of the vacuolar apparatus. I n Lysosomes in biology and pathology. Frontiers of biology series: 14B. Edited by J. T. Dingle and H. B. Fell. North-Holland Publishing Company, Amsterdam and London. pp. 64-1 12. DE DUVE,C., and R. WATTIAUX. 1966. Functions of lysosomes. Annu. Rev. Physiol. 28: 435-492. DOUGHRI, A. M., and K. P. ALTERA.1973. Host cell range of chlamydial infection in the neonatal bovine gut. J . Comp. Pathol. 83: 107-114. DOUGHRI, A. M., J . STORZ,and K. P. ALTERA.1972. Mode of entry and release of chlamydiae in infections of intestinal epithelial cells. J. Infect. Dis. 126: 652-657. DOUGHRI, A. M., J. STORZ,and A. K. EUGSTER. 1973. Ultrastructural changes in the Chlanlydia-infected ileal mucosa of newborn calves. Vet. Pathol. 10: 114-123. DROBYSHEVSKAYA, A. R., V. E. PIGAREVSKY, and A. A. SMORODINTSEV. 1%2. Activity of phagocytic factors in experimental infection of white mice with mouse pneumonia and meningopneumonitis viruses. Acta Virol. 6: 458-470. FORSBERG, C. W., M. K. RAYMAN, J . W. COSTERTON, and R. A. MACLEOD.1972. Isolation, characterization and ultrastructure of the peptidoglycan layer of a marine pseudomonad. J. Bacteriol. 109: 895-905. FREEMAN, B. A., and B. H. RUMACK. 1%4. Cytopathogenic effect of Brucella spheroplasts on monocytes in tissue culture. J . Bacteriol. 88: 1310-1315. H A T ~ E NB., A,, and S. E. SULKIN. 1%6. Intracellular production of Brucella L forms. 11. Induction and survival of Brucella abortus L forms in tissue culture. J. Bacteriol. 91: 14-20. KONDO,L., L. HANNA,and H. KESHISHYAN. 1973. Reduction in chlamydial infectivity by Iysozyme. Proc. Soc. Exp. Biol. Med. 142: 131-132.
and J . C. WILT. 20. KORDOVA,N., J . HOOGSTRATEN, 1973. Lysosomes and the "toxicity" of Rickettsiales. IV. Ultrastructural studies of macrophages infected with a cytopathic L cell-grown C.psittaci 6BC strain. Can. J. Microbiol. 19: 315-320. 21. KORDOVA,N., C. MARTIN,J. C. WILT, and W. MEYER. 1973. Immunofluorescence of peritoneal phagocytes after infection in vivo with egg-attenuated Chlamydia psittaci 6BC. Can. J. Microbiol. 19: 1425-1429. 22. KORDOVA,N., J. C. WILT, and C. MARTIN.1975. Lysosomes and the "toxicity" of Rickettsias. VI. In vivo response of mouse peritoneal phagocytes to L-cell-grown Chlamydia psittaci 6BC strain. Can. J. Microbiol. 21: 323-331. 23. KORDOVA,N., J . C. WILT, and L. POFFENROTH. 1973. Lysosomes and the "toxicity" of Rickettsiales. V. In vivo relationship of peritoneal phagocytes and egg-attenuated C. psittaci 6BC. Can. J . Microbiol. 19: 1417-1423. and W. 24. KORDOVA,N., J. C. WILT, E. SHEWCHUK, MEYER. 1975. Immunofluorescence of peritoneal phagocytes after infection of mice with L-cellattenuated Chlamydia psittaci 6BC. Can. J. Microbiol. 21: 759-763. 25. LEPINAY,A,, J . ORFILA, A. ANTEUNIS,J . M. BOUTRY,L. ORME-ROSELLI, and R. ROBINEAUX. 1970. Etude en microscopie electronique du developpement et de la morphologie de Chlamydia psittaci dans les macrophages de souris. Ann. Inst. Pasteur (Paris). 119: 223-231. 26. LEPINAY,A., R. ROBINEAUX, J . ORFILA, L. ORME-ROSSELLI, and J . M. BOUTRY. 1971. Ultrastructure et cytochimie ultrastructurale des membranes de ~ h l a m ~ dpsittaci. ia Arch. Gesamte Virusforsch. 33: 271-280. LITWIN,J. 1959. The growth cycle of the psittacosis group of organisms. J. Infect. Dis. 105: 129-160. MCDERMOTT, W. 1958. Microbial persistence. Yale J. Biol. Med. 30: 257-291. MCGHEE,J . R., and B. A. FREEMAN. 1970. Effect of lysosomal enzymes on Br~rcella. Reticuloendothel. SOC.8: 208-219. MANIRE, G. P., and A. TAMURA. 1967. Preparation and chemical composition of the cell walls of mature infectious dense forms of meningopneumonitis organisms. J. Bacteriol. 94: 1178-1 183. MITSUI,Y., M. FUJIMOTO, and M. KAIIMA.1%4. Development and morphology of trachoma agent in the yolk sac as revealed by electron microscopy. Virology, 23: 30-45. PEARSALL, N. N., and R. S . WEISEN.1970. The macrophage. Lea and Febiger, Publishers, Philadelphia, Pa. N. KORDOVA, POFFENROTH, L., J . W. COSTERTON, and J. C. WILT. 1973. Ultrastructural studies of Chlanlydia psittaci 6BC variant strains. I. Ultrastructure of the surface layers of egg-passaged 6BC strain. Can. J. Microbiol. 19: 887-894. 1%3. Purification and TAMURA, A., and N. HIGASHI. chemical composition of meningopneumonitis virus. Virology, 20: 596-604. TAMURA, A., and G. P. MANIRE. 1974. Hemagglutinin in cell walls of Chlamydia psittaci. J . Bacteriol. 118(1): 144-148.
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36. T A M U R AA,, , A. M A T S U M O T O N., ~HIGASHI. ~~ 1967. Purification and chemical composition of reticulate bodies of the meningopneumonitis organisms. J. Bacteriol. 93: 2003-2008. 37. T A M U R AA,, , A. MATSUMOTO, G . P. MANIRE, and N. HIGASHI.1971. Electron microscopic observations on the structure of the envelopes of mature elementary bodies and developmental reticulate forms of Chlarnydiapsitfaci. J. Bacterial. 105: 355-360.
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38. WEISSMANN, G . 1967. The role of lysosomes in inflammation and d~sease. Annu. Rev. Med. 18: 97-1 12. 39. WEISSMANN, G., P. DUKOR,and R. B. ZURIER.1971. Effect of cyclic AMP on release of lysosomal enzymes from phagocytes. Nat. New Biol. 231: 131-135.
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NONNAKORDOVP;,JOHNC. WILT, LYNN BURTON,A N D CAROLINE MARTIN Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba Accepted February 21, 1975
KORDOVL, N., J. C. WILT,L. BURTON,and C. MARTIN.1975. Electron microscopy of the in vivo internalization of virulent Chlamydia psittaci 6BC strain. Can. J . Microbiol. 21: 945-953. The internalization of virulent Chlamydia psittaci 6BC particles by wandering mononuclear phagocytes in the peritoneal cavity of intraperitoneally inoculated mice occurred asynchronously, i.e., fragile reticulate bodies (RB) appeared to be more readily phagocytized than the rigid elementary bodies (EB). Early damage of mononuclear phagocytes occurred after internalization of chlamydiae. This was followed by a decreased uptake of particles, and may explain the relatively long persistence (up to 6 h after inoculation) of free, extracellular, "swollen," and RB-like particles. Internalized particles within phagolysosomes showed varying degrees of disintegration. The subsequent influx of polymorphonuclear phagocytes and monocytes into the inflammed peritoneal cavity may explain the rapid disappearance of chlamydiae and their antigens from the peritoneal fluid. The alteration in ultrastructure of peritoneal cells and chlamydial parasites during the inflammatory process are discussed. KORDOVL, N., J. C. WILT,L. BURTONet C. MARTIN.1975. Electron microscopy of the in vivo internalization of virulent Chlamydia psittaci 6BC strain. Can. J. Microbiol. 21: 945-953. Chez des souris inoculees par voie intrapkritoneale le passage des particules virulentes de Cl~lamydiapsittaci 6BC B I'interieur des mononucleaires circulants dans la cavitC peritontale se fait de fagon asynchrone. Les corps rtticulks fragiles (RB) semblent en effet phagocytes plus rapidement que les corps eltmentaires rigides (EB). Aprks la penetration des chlamydies, les phagocytes mononuclCaires sont rapidement endommagks. Cela entraine une diminution de la captation des particules et peut expliquer la persistance relativement longue (jusqu'a 6 h aprks I'inoculation) de particules extracellulaires "gonflCes" et d'allure RB. Les particules intracellulaires incluses dans des phagolysosomes sont dksintegrees a differents degrCs. L'arrivee subskquente des polynucleaires et des monocytes dans la cavitt peritoneale inflammee peut expliquer la disparition rapide dans le liquide peritoneal des chlamydies et de leurs antigknes. On discute de I'altCration de I'ultrastructure des cellules pCritonCales et des parasites chlamydiaux au cours du processus inflammatoire. [Traduit par le journal]
Introduction contrast, after infection of mice with an aviruIn our previous in vivo studies of chlamydiae lent 6BC strain readily identifiable parasites the response of wandering mouse peritoneal were observed in mononuclear phagocytes. The phagocytes and the fate of either a virulent or lysosomes of macrophages were refractory to avirulent (for mouse) Chlamydia psittaci 6BC avirulent 6BC particles that were initially instrain has been followed with cytochemical gested and some macrophages were "transmethods by light microscopy (22, 23) and im- formed" into large epithelioid cells containing munofluorescence (21, 24). We described the chlamydial inclusions in the cytoplasm (23). The objective of the present study was to virulent 6BC strain (grown in L cells) as inducing observe the relationship between parasites and an early injurious effect on mononuclear phagol wandering phagocytes at the ultrastructural cytes and their lysosomes; a marked influx of polymorphonuclear phagocytes (PMN's) oc- level during the early crucial phases after intracurred which showed early karyorrhexis and peritoneal inoculation of mice with virulent 6BC lysis. GimCnez or Giemsa staining failed to detect chlamydiae in peritoneal fluids up to 6 Material and Methods days after inoculation of mice. Chlamydiae The C . psittaci 6BC strain (grown in L cells), the CF-1 were recoverable from the peritoneal fluid only strain of mice, and the procedures of inoculation and during the 1st h after inoculation of mice. In harvesting peritoneal cells used throughout these studies 'Received August 29, 1974.
were the same a s described in the preceding paper (22). For ultrastructural studies, samples of semipurified
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chlamydiae and peritoneal cells from infected and control mice were processed by methods described by others (16) and used in our previous studies (20, 33). Mice were injected intraperitoneally with 0,2 ml of semipurified chlamydial inoculum as described before (22) and killed at medetermined time intervals after inoculation. Three miililitres of fresh 1% glutaraldehyde (Ladd Research Industries) in phosphate buffer at a pH of 7.0 was injected into the peritoneal cavity, the fluid was withdrawn and centrifuged at 1000 rpm for 20 min, and the pellet was processed for electron microscopy. Thin sections were obtained using a LKB ultramicrotome with a glass knife, picked up on 400-mesh copper specimen grids, and double-stained with uranyl acetate and lead citrate. A Philips 201 electron microscope was used to examine the material. Micrographs were made at magnifications up to 8640 diameters and then enlarged photographically. Other methods are described along with Results.
Results Differential counts of peritoneal cells after inoculation of mice with virulent 6BC chlamydiae have shown that few PMN's were present in the peritoneal cavity up to 6 h after inoculation. The early events of the interaction are therefore predominantly concerned with mononuclear phagocytes. The polymorphic chlamydial particles (5) present in thin sections (Figs. 9 and 10) were differentiated in accordance with others (5,25,26) into three main forms: small (0.25-0.35 microns (p) in diameter), dense, centered, rigid, elementary bodies (EB), large, (0.5 p) nonrigid, reticulate bodies (RB), and intermediate (0.35-5 p) bodies (IB). The particles were identified in their relation to phagocytes as completely unattached, as present in invaginations, as attached, or as intracellular. As early as 5 min after intraperitoneal inoculation of chlamydiae, RB and IB were seen within phagocytes surrounded by an intactappearing cellular membrane (Fig. 1) most probably derived from the surface membrane of the phagocyte. Several EB appeared to be attached to phagocytes (Fig. 1). At 5 min most of the organisms were unattached (Fig. 11). This is in agreement with assays of chlamydial infec-
tivity performed in parallel in which high titers of infective parasites were found in the fractions of peritoneal fluids collected 5 rnin after inoculation (22). At 10 rnin after inoculation there was a marked increase in the number of EB and IB attached to cells (Fig. 3). Internalized EB and IB showing varying degrees of ultrastructural alterations were localized in different compartments in the cytoplasm of the phagocytes with an average of three to eight particles per phagocyte profile (Figs. 4 and 8). Large, well-defined, internalized RB were rarely detected at 10 rnin or later after inoculation although they were seen extracellularly (Figs. 12 and 13). In some cells particles appeared to be closely surrounded by a cellular membrane (Fig. 2), in some the particles were present singly or in clusters in large vacuoles, and in other cells the particles were detected in dense cytoplasmic areas with no sharp boundary between them and the surrounding cytoplasm (Fig. 8). Most phagocytes with or without chlamydiae exhibited alterations of the lysosomal apparatus (10, 11) including incomplete or fragmentary vesicular membranes; some vesicles were empty and others contained granular material or dense bodies (Fig. 8). Despite the increased uptake of particles, collections of unattached chlamydial particles remained in the fluid interspersed amongst dense granular material, small empty vesicles, residual bodies, fragments of membranes, and lamellar structures (Figs. 5 and 6). Extracellular particles harvested from peritoneal fluids at predetermined times after inoculation appeared agglutinated (Fig. 12) although they were dispersed in the inoculum before inoculation. In contrast to the relatively large number of EB inside phagocytes at 10 rnin after inoculation, few EB could be detected in phagocytes 30 rnin after inoculation. Particles were often located in large peripheral vacuoles, most probably invaginations of the outer cytoplasmic membrane (Fig. 5). Many unattached EB were also
ABBREVIATIONS USED: MP, mononuclear phagocyte; M, mitochondrion; N, nucleus; L, lysosome. The bar on all micrographs represents 0.1 p. FIG. 1. Internalized RB in MP 5 min after inoculation of mice. Fig. 2. Internalized IB and EB 10 min after inoculation of mice. FIG. 3. EB appear attached to the outer membrane of M P 10 min after inoculation. FIG. 4. EB (arrows) are entrapped between microvilli of MP. There is some variation in the density of the cytoplasm of EB enclosed by two closely apposed double-track structures. FIGS.5-6. Localization of EB and different ultrastructural alterations of EB and MP 30 rnin after inoculation. FIG. 7. Disintegration of EB (arrows) in phagolysosomes of M P 6 h after inoculation.
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FIG.8. Fate of EB (arrows) in different compartments of MP 10 min after inoculation.
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FIGS.9-10, EB and RB in semipurified suspension before inoculation. FIGS.11-14. Alterations in the ultrastructure of extracellular chlamydia1 particles in peritoneal fluid; FIGS.1 1 and 12, 5 min after inoculation; FIG. 13, 10 min after inoculation: and FIG. 14, 1 h after inoculation.
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seen interspersed between phagocytes (Fig. 6). Most phagocytes showed ultrastructural alterations such as large perinuclear and peripheral vacuolization, paucity or alteration of cytoplasmic organelles, dense and granular cytoplasmic areas, thinning of ground cytoplasm, and complete cellular disintegration. Peritoneal fluid collected at 1 and 6 h after inoculation required extensive screening to detect a few severely damaged intracellular particles (Fig. 7). It is noteworthy that extracellular accumulations of agglutinated, severely altered particles sometimes entrapped in whorls of membranes were detected on several occasions. The ultrastructural alteration of particles undergoing disintegration in phagolysosomes is seen in Figs. 7 and 8. Many cell-free particles recovered from peritoneal fluids at 1 and 6 h appeared "swollen" or "fuzzy" and RB-like (Fig. 14); their surface envelopes appeared loosened or to be undergoing dissolution. In some particles retraction and in others rarefication of the cytoplasm was seen. No chlamydiae or a few chlamydiae-like structures were seen in peritoneal fluids collected at day 1 and up to 6 days after inoculation, i.e. at a time when the marked influx of PMN's occurred in the inflammed peritoneum (22). In contrast to electron microscopic observations, the greatest amount of cell-bound chlamydial infectivity was detected at 1 h after inoculation. No infectivity was identified in the cell-free fraction of peritoneal fluids at this time. No infectivity was identified in either cell-bound or cell-free fractions sampled at 6 h, 1 day, and daily up to 6 days after inoculation. The observations as described above have shown that the internalization of virulent 6BC chlamydial particles by wandering mononuclear phagocytes in the peritoneal cavity of intraperitoneally inoculated mice occurred asynchronously. Fragile RB and IB appeared to be more readily phagocytized than the rigid EB. After internalization of chlamydiae early damage of mononuclear phagocytes occurred. This apparently caused a decreased uptake of particles and may explain the relatively long persistence (up to 6 h after inoculation) of unattached extracellular particles. Internalized particles were disintegrated in phagolysosomes. The increased influx of PMN's and monocytes from the circulation into the inflammed peritoneal cavity might
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explain the subsequent rapid disappearance of chlamydial particles and their antigens (22, 24) from the peritoneal fluid examined later, up to 6 days after inoculation.
Discussion The early chlamydiae-phagocyte interaction in vivo has not been analyzed by electron microscopy before the present study. Some in vivo ultrastructural studies during the later stages of infection have been described in connection with intestinal chlamydial infections of calves (12, 13, 14) and intraarticular infection of lambs (9) in which the authors observed a varying response of macrophages to chlamydiae. Our studies have demonstrated that the strain of chlamydiae and its virulence for the mouse strongly affect the outcome of the chlamydiae-phagocyte relationship (22, 23). After inoculation of virulent 6BC chlamydiae an early ultrastructural alteration developed in both mononuclear phagocytes and in the parasites, with subsequent rapid loss of the chlamydial infectivity. In an earlier study Drobyshevskaya and co-workers (15) demonstrated a rapid inactivation of chlamydial infectivity in the peritoneal cavity of mice using two virulent chlamydial strains, but electron microscopy was not performed. Electron microscopy has revealed several patterns of response of phagocytes to microbes depending on the virulence of the parasite and on the degree of resistance of the phagocyte (1,2, 3, 32). A frequent response to the uptake of microbes is the formation of phagolysosomes with the subsequent digestion of microbes. This pattern has been described in mouse peritoneal macrophages and in human macrophages in several bacterial infections (1, 32). Less frequently virulent, toxic microbes affect the integrity of the phagocyte membrane (1, 2, 32). It is not clear how lysosomes injure the cell of which they constitute a part or how they affect surrounding tissues (I I). It appears that under some circumstances direct injury to lysosomes induces damage to cellular structures leading to eventual cell death. Release of enzymes from disrupted lysosomes through the cell membrane by a kind of exocytosis has been suggested as one of several possible mechanisms for damage of surrounding structures. Evidence has also been obtained for the presence of several tissue-
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damaging substances especially in leucocyte lysosomes (1 1, 38, 39). Because of the agglutinogens of chlamydial EB (6, 35) one might suggest that their injurious action upon phagocytes may be initiated on the surface of certain phagocytes. If this indeed is the case, the possibility exists that at least in some phagocytes, EB, instead of being engulfed, may be eluted because of the inhibited phagocytic activity of such injured phagocytes. Furthermore, injury of the cell surface per se may elicit release of lysosomal enzymes inside and outside the cell (2, 11). One can therefore not dismiss the possibility that the structure and function of the chlamydial EB may be altered during adsorption to some phagocytes and before subsequent uptake by other phagocytes. It is possible to speculate that the accumulation of cell-free ultrastructurally damaged RB-like chlamydial particles in the peritoneal fluid could be attributed to the early damage of the surface of the phagocyte membrane and release of lysosoma1 enzymes as well as tissue-damaging substances from these cells. Lysozyme is usually associated with the lysosomal granule of host cells, particularly in phagocytes (8). Lysosomal extracts and lysosomal enzymes such as lysozyme, leucozyme, acid phosphatase, and phospholipase have produced fragile forms from gram-positive as well gram-negative bacteria (4, 29). Macrophages have been reported to exert a lysozyme-like activity on Brucella (29); lysosomal extracts from PMN's converted smooth Brucella to spheroplasts and killed artificially induced spheroplasts in vitro. Egg white lysozyme has not elicited this reaction, thus indicating the presence of anti-Brucella substances in phagocyte lysosomes (29). In this connection it is noteworthy that a limited degree of chlamydial infectivity has been reported in chlamydiaeinfected tissue cultures after treatment with chicken egg white lysozyme (19). It is generally accepted as an oversimplification that chlamydial populations consist of two kinds of particles; the infectious, rigid EB which reorganizes after phagocytosis and during the "development cycle" into noninfectious, fragile RB. The mechanism whereby the small infectious EB transforms intracellularly into the large noninfectious cell wall deficient form and back to the small form again is largely unknown. In the present studies fragile RB and (or) IB
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appeared to be more readily phagocytized by mononuclear phagocytes than the rigid EB. This raises several questions. Whatever the nature of RB, evidence is accumulating (7, 30, 34, 36, 37) that the surface envelopes of fragile RB differ physically and chemically from that of the rigid EB. It has been shown recently (35) that hemagglutinogen (6) present in high titers in homogenates of EB and their cell walls was almost absent in preparation of purified RB. The fragility of RB has been attributed to a lack of the rigidity factor in the surface envelopes of these large particles (37). Whatever the basis of the cell wall deficiency of RB may be, it is now well established that in general the behavior of cell wall deficient microbes in vivo is different from that of their original rigid classical forms. Some cell wall deficient variants are for example "indifferent" to certain antimicrobial and host tissue factors (28). Furthermore, some cell wall deficient bacterial variants are rather rapidly phagocytized (17) and also survive within macrophages and monocytes (1 8,32). It has been demonstrated (32) that simultaneous exposure of macrophages to more than one kind of particle can influence phagocytosis and also clearance by these cells. If indeed the fragile RB are more rapidly phagocytized than the rigid EB in certain cells, the possibility exists that in a mixed population of chlamydial strains the RB ingested initially could interfere with the subsequent uptake of EB. Electron microscopic studies have demonstrated that the proportion of RB in the overall population of chlamydiae varies in different hosts (27, 31) but the reasons for these population changes are unknown.
Acknowledgments This research was supported by a Medical Research Council grant No. MA3901. Appreciation is expressed to Mrs. Eleanor Shewchuk and Mrs. Esther Elias for assistance. 1. ALLEN,J . M. 1969. Lysosomes in bacterial infection. In Lysosomes in biology and pathology. Frontiers of biology series: 14B. Edited b y J. T . Dingle and H. B. Fell. North-Holland Publishing Company, Amsterdam and London. pp. 41-68. A. 1967. Lysosomes in virus-infected cells. 2. ALLISON, In Perspectives in virology. V. Virus directed host response. Edited b y V. POLLARD.Academic Press, Inc., New York and London. pp. 29-60. 3. ALLISON, A. C., and L. MALLUCCI. 1%5. Histochemical studies of lysosomes and lysosomal enzymes in
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and J . C. WILT. 20. KORDOVA,N., J . HOOGSTRATEN, 1973. Lysosomes and the "toxicity" of Rickettsiales. IV. Ultrastructural studies of macrophages infected with a cytopathic L cell-grown C.psittaci 6BC strain. Can. J. Microbiol. 19: 315-320. 21. KORDOVA,N., C. MARTIN,J. C. WILT, and W. MEYER. 1973. Immunofluorescence of peritoneal phagocytes after infection in vivo with egg-attenuated Chlamydia psittaci 6BC. Can. J. Microbiol. 19: 1425-1429. 22. KORDOVA,N., J. C. WILT, and C. MARTIN.1975. Lysosomes and the "toxicity" of Rickettsias. VI. In vivo response of mouse peritoneal phagocytes to L-cell-grown Chlamydia psittaci 6BC strain. Can. J. Microbiol. 21: 323-331. 23. KORDOVA,N., J . C. WILT, and L. POFFENROTH. 1973. Lysosomes and the "toxicity" of Rickettsiales. V. In vivo relationship of peritoneal phagocytes and egg-attenuated C. psittaci 6BC. Can. J . Microbiol. 19: 1417-1423. and W. 24. KORDOVA,N., J. C. WILT, E. SHEWCHUK, MEYER. 1975. Immunofluorescence of peritoneal phagocytes after infection of mice with L-cellattenuated Chlamydia psittaci 6BC. Can. J. Microbiol. 21: 759-763. 25. LEPINAY,A,, J . ORFILA, A. ANTEUNIS,J . M. BOUTRY,L. ORME-ROSELLI, and R. ROBINEAUX. 1970. Etude en microscopie electronique du developpement et de la morphologie de Chlamydia psittaci dans les macrophages de souris. Ann. Inst. Pasteur (Paris). 119: 223-231. 26. LEPINAY,A., R. ROBINEAUX, J . ORFILA, L. ORME-ROSSELLI, and J . M. BOUTRY. 1971. Ultrastructure et cytochimie ultrastructurale des membranes de ~ h l a m ~ dpsittaci. ia Arch. Gesamte Virusforsch. 33: 271-280. LITWIN,J. 1959. The growth cycle of the psittacosis group of organisms. J. Infect. Dis. 105: 129-160. MCDERMOTT, W. 1958. Microbial persistence. Yale J. Biol. Med. 30: 257-291. MCGHEE,J . R., and B. A. FREEMAN. 1970. Effect of lysosomal enzymes on Br~rcella. Reticuloendothel. SOC.8: 208-219. MANIRE, G. P., and A. TAMURA. 1967. Preparation and chemical composition of the cell walls of mature infectious dense forms of meningopneumonitis organisms. J. Bacteriol. 94: 1178-1 183. MITSUI,Y., M. FUJIMOTO, and M. KAIIMA.1%4. Development and morphology of trachoma agent in the yolk sac as revealed by electron microscopy. Virology, 23: 30-45. PEARSALL, N. N., and R. S . WEISEN.1970. The macrophage. Lea and Febiger, Publishers, Philadelphia, Pa. N. KORDOVA, POFFENROTH, L., J . W. COSTERTON, and J. C. WILT. 1973. Ultrastructural studies of Chlanlydia psittaci 6BC variant strains. I. Ultrastructure of the surface layers of egg-passaged 6BC strain. Can. J. Microbiol. 19: 887-894. 1%3. Purification and TAMURA, A., and N. HIGASHI. chemical composition of meningopneumonitis virus. Virology, 20: 596-604. TAMURA, A., and G. P. MANIRE. 1974. Hemagglutinin in cell walls of Chlamydia psittaci. J . Bacteriol. 118(1): 144-148.
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