Vol. 22, No. 2

INFECTION AND IMMUNITY, Nov. 1978, P. 620-622 0019-9567/78/0022-0620$02.00/0 Copyright © 1978 American Society for Microbiology

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Response of C3H/HeJ and C3H/HeN Mice and Their Peritoneal Macrophages to the Toxicity of Chlamydia psittaci Elementary Bodies B. E. IVINS* AND P. B. WYRICK Department of Bacteriology and Immunology, The University of North Carolina School of Medicine, Chapel Hill, North Carolina 27514

Received for publication 28 July 1978

Intravenous injection of toxic doses of Chlamydia psittaci elementary bodies into endotoxin-responsive C3H/HeN mice or endotoxin-nonresponsive C3H/HeJ mice resulted in essentially identical time intervals to death. Inoculation of monolayer cultures of thioglycolate-stimulated peritoneal macrophages from the two strains of mice with 250 elementary bodies per macrophage resulted in immediate host cell toxicity, although the C3H/HeJ macrophages were somewhat less sensitive to elementary body toxicity than were the C3H/HeN macrophages.

C3H/HeN mice respond normally to endotoxin, whereas the closely genetically related C3H/HeJ strain of mice (3, 14, 15, 20) and their B cells (13, 14, 16, 20) and macrophages (6, 13) express a markedly reduced capacity to respond to the effects of endotoxic lipopolysaccharide (LPS). A biological system is thus available for determining the presence of endotoxic LPS in a particular microbial organism. Investigators studying Chlamydia sp. have demonstrated the similarity in biochemical composition of chlamydial elementary body (EB) walls to the walls of gram-negative bacteria (7,9) and have identified a lipid-polysaccharide-protein complex responsible for chlamydial group antigenicity (1, 4, 5, 11). Dhir et al. (4) also demonstrated the presence of a 2-keto-3-deoxy sugar in the polysaccharide fraction from the group antigen which was similar, although not identical, to the 2keto-3-deoxyoctanoic acid in Salmonella enteritidis LPS. Such findings suggest the possibility of a chlamydial endotoxin. The studies described in this report deal with the interaction of Chlamydia psittaci EB with normal female mice of both the C3H/HeN strain (Division of Research Services, National Institutes of Health, Bethesda, Md.) and the C3H/HeJ strain (Jackson Laboratories, Bar Harbor, Maine) and their thioglycolate-stimulated peritoneal macrophages. The Cal 10 meningopneumonitis strain of C. psittaci used in these investigations was propagated, harvested, purified, and titrated for infectivity as previously described (21, 22). In the in vivo studies, C3H/HeN and C3H/HeJ mice, in groups of five, were injected intravenously with EB in 0.1% sodium carbonate 620

(0.2 ml/mouse). The time interval to death for each animal was recorded, and the mean time interval to death for each group of mice was determined. In each of two separate experiments (Table 1), endotoxin-responsive, as well as endotoxin-nonresponsive, mice succumbed to toxic death, and there was no significant difference in the time interval to death between the two mouse strains. Control animals receiving injections of carbonate alone exhibited no pathological symptoms. These data suggest that the in vivo toxicity of the Cal 10 meningopneumonitis strain of C. psittaci EB is probably not a function of endotoxic LPS in the infectious agent. This is consistent with previous reports which noted several characteristics of chlamydial toxicity quite atypical of gram-negative endotoxicity, including heat lability (10, 12, 17, 18, 22) and the ability to form a toxoid with Formalin (8, 12). Interestingly, the antigenicity of the putative chlamydial toxic factor is quite heat stable (19). In the in vitro studies, the toxicity of EB for thioglycolate-stimulated, cultured mouse peritoneal macrophages was assayed as previously described (22). EB were inoculated onto monolayers of macrophages, 5 x 10' per 35-mm plastic culture dish, at a multiplicity of infection of 250:1. The release of lactate dehydrogenase (LDH) into the culture medium was monitored as an index of macrophage damage (2). The results of four independent experiments (Fig. 1) demonstrate that macrophages from both strains of mice exhibited immediate cytotoxicity to the EB, although the level of the cytotoxic response in the C3H/HeN mice was consistently slightly greater (less than twofold) than that in the C3H/HeJ macrophages. Furthermore, the

VOL. 22, 1978


TABLE 1. Toxicity of C. psittaci EB for endotoxinresponsive C3H/HeN and endotoxin-nonresponsive C3H/HeJ mice Mean time to death (h)a Group of mice Expt lb

Expt 2c

9.6 (8.8-10.0) 4.7 (4.0-5.2) C3H/HeN 10.0 (9.0-11.3) 5.4 (4.2-6.5) C3H/HeJ 'Five mice per group received injections of EB. The range of values for each group is given within parentheses. b 5.5-week-old mice, approximately 15.5 g, received 5 x 109 EB (0.68 inclusion-forming unit per particle) per mouse.

e 3.5-week-old mice, approximately 13.5 g, received 1.4 x 109 EB (0.22 inclusion-forming unit per particle) per mouse.

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and d) compared with the release of LDH from macrophages inoculated with the more infective EB (Fig. la and c). Other researchers have reported similar findings with chlamydiae linking viability with toxicity (10, 12). Although the difference in LDH release between the macrophages of the two mouse strains was relatively slight, at least a partial role for chlamydial LPS in the in vitro cytotoxic response cannot be ruled out. Indeed, Glode et al. (6) have demonstrated for certain preparations of endotoxin that C3H/HeN peritoneal macrophages exhibit only a two- to threefold-greater cytotoxic response than C3H/HeJ macrophages. The in vitro toxicity of chlamydiae for L cells (10) and for macrophages (17, 22) is abolished by relatively mild heating at 600C for 3 to 5 min. This would seem to indicate that chlamydial cytotoxicity for macrophages is not LPS related. However, although the heat stability of endotoxin with respect to its in vivo toxicity is well documented, the effect of heat on LPS in vitro toxicity for macrophages is unknown. Furthermore, the in vitro-cultured macrophage is an isolated cell in an artificial environment, and a strict a priori correlation with the in vivo situation is not tenable. Therefore, definitive conclusions cannot yet be drawn concerning the involvement of LPS in chlamydial macrophage cytotoxicity.





1. Benedict, A. A., and E. O'Brien. 1956. Antigenic studies on the psittacosis-lymphogranuloma venereum group of viruses. I. Characterization of comnplement-fixing antigens extracted with sodium lauryl sulfate. J. Immunol.


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FIG. 1. Release of LDH from C3H/HeN (0) and C3H/HeJ (0) mouse peritoneal macrophages inoculated with C. psittaci EB at a multiplicity of 250 EB/macrophage. Uninoculated C3H/HeN (0) and C3H/HeJ (U macrophages showed little release of LDH into the menstrum. (a) 6.5-week-old mice, 0.375 inclusion-forming unit (IFU) per EB; (b) 10.5-weekold mice, 0.22 IFUIEB; (c) 13.0-week-old mice, 0.299 IFUIEB; (d) 14.5-week-old mice, 0.183 IFUlparticle. The range of values is denoted by the brackets. Each mean value represents the average LDH release in four identical plates.

EB-induced cytotoxic response was related to viability of the chlamydiae, as evidenced by the decreased release of LDH from macrophages inoculated with EB of lower infectivity (Fig. lb

2. Bergmeyer, H. U. 1963. Methods of enzymatic analysis, p. 736-741. Academic Press Inc., New York. 3. Chedid, L, M. Parant, C. Damais, F. Parant, D. Juy, and A. Galelli. 1975. Failure of endotoxin to increase nonspecific resistance to infection of lipopolysaccharide low-responder mice. Infect. Immun. 13:722-727. 4. Dhir, S. P., S. Hakomori, G. E. Kenny, and J. T. Grayston. 1972. Immunochemical studies on chlamydial group antigens. J. Immunol. 109:116-122. 5. Dhir, S. P., G. E. Kenny, and J. T. Grayston. 1971.

Characterization of the group antigen of Chlamydia trachomatis. Infect. Immun. 4:725-730. 6. Glode, L M., A. Jaques, S. E. Mergenhagen, and D. L Rosenstreich. 1977. Resistance of macrophages from C3H/HeJ mice to the in vitro cytotoxic effects of endotoxin. J. Immunol. 119:162-166. 7. Jenkin, H. M. 1960. Preparation and properties of cell walls of the agent of meningopneumonitis. J. Bacteriol. 80:639-647. 8. Manire, G. P., and K. F. Meyer. 1950. The toxins of the psittacosis-lymphogranuloma group of agents. H. Effects of aureomycin and penicillin upon the toxins of psittacosis viruses. J. Infect. Dis. 86:233-240. 9. 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. 94:1178-1183. Hatch, G. I. Byrne, and K. R.

10. Moulder, J. W., T. P.








Kellogg. 1976. Immediate toxicity of high multiplicities of Chiamydia psittaci for mouse fibroblasts (L cells): Infect. Immun. 14:277-289. Narita, T., and G. P. Manire. 1976. Protein-carbohydrate-lipid complex isolated from cell envelopes of Chlamydia psittaci in alkaline buffer and ethylenediaminetetraacetate. J. Bacteriol. 125:308-316. Rake, G., and H. Jones. 1943. Studies on lymphogranuloma venereum. II. The association of specific toxins with agents of the lymphogranuloma-psittacosis group. J. Exp. Med. 79:463-485. Rosenstreich, D. L., L. M. Glode, L. M. Wahl, A. L Sandberg, and S. E. Mergenhagen. 1977. Analysis of the cellular defects of endotoxin-unresponsive C3H/HeJ mice, p. 314-320. In D. Schlessinger (ed.), Microbiology-1977. American Society for Microbiology, Washington, D.C. Skidmore, B. J., J. M. Chiller, D. C. Morrison, and W. 0. Weigle. 1975. Immunologic properties of bacterial lipopolysaccharide (LPS): correlation between the mitogenic, adjuvant and immunogenic activities. J. Immunol. 114:770-775. Sultzer, B. M. 1968. Genetic control of leucocyte responses to endotoxin. Nature (London) 219:1253-1254.

INFECT. IMMUN. 16. Sultzer, B. M., and B. S. Nilsson. 1972. PPD tuberculin-a B cell mitogen. Nature (London) New Biol. 240:198-200. 17. Taverne, J., W. A. Blyth, and R. C. Ballard. 1974. Interactions of TRIC agents with macrophages: effects on lysosomal enzymes of the cell. J. Hyg. 72:297-309. 18. Wang, S. P., and J. T. Grayston. 1967. A potency test for trachoma vaccine using the mouse toxicity prevention test. Am. J. Ophthalmol. 63:1443-1454. 19. Wang, S. P., G. E. Kenny, and J. T. Grayston. 1967. Characterization of trachoma antigens protective against mouse toxicity. Am. J. Ophthalmol. 63: 1454-1461. 20. Watson, J., and R. Riblet. 1974. Genetic control of responses to bacterial lipopolysaccharides in mice. I. Evidence for a single gene that influences mitogenic and immunogenic responses to lipopolysaccharides. J. Exp. Med. 140:1147-1160. 21. Wyrick, P. B., and E. A. Brownridge. 1978. Growth of Chiamydia psittaci in macrophages. Infect. Immun. 19:1054-1060. 22. Wyrick, P. B., E. A. Brownridge, and B. E. Ivins. 1978. Interactions of Chlamydia psittaci with mouse peritoneal macrophages. Infect. Immun. 19:1061-1067.

HeN mice and their peritoneal macrophages to the toxicity of Chlamydia psittaci elementary bodies.

Vol. 22, No. 2 INFECTION AND IMMUNITY, Nov. 1978, P. 620-622 0019-9567/78/0022-0620$02.00/0 Copyright © 1978 American Society for Microbiology Print...
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