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Immunofluorescence of peritoneal phagocytes after infection of mice with L-cell-attenuatedChlamydia psittaci 6BC1 NONNAKORDOVA, JOHNC. WILT, ELEANOR SHEWCHUK, A N D WALLYMEYER Depnrtmer~tof Medico1 Microbiology, Urliversity of Mnnitobn, Wirzr~ipeg, Mnnitobn

Accepted February 6, 1975 KonoovA, N . , J . C. WILT, E. SHEWCHUK, and W. MEYER.1975. Irnrnunofluorescence of peritoneal phagocytes after infection of mice with L-cell-attenuated Chlor,lyrlicr psittrrci 6BC. Can. J. Microbiol. 21: 759-763. Large amounts of particulate antigen ofClrlnmydiapsittnci 6BC attenuated by growth in Lcells were phagocytized by peritoneal mononuclear phagocytes during the 1st h after intraperitoneal inoculation. The phagocytes subsequently destroyed the irnmunofluorescent (IF) properties of the chlamydial antigens. It is suggested that the early damage of phagocytes by lysosornal enzymes activation induced by chlamydiae contributed to the relatively early disappearance of IF antigens from the peritoneal fluids. KORDOVL,N . , J. C. WILT, E. S H E W C H Uet K W. MEYER.1975. Immunofluorescence of peritoneal phagocytes after infection of mice with L-cell-attenuated C l r l ~ r ~ ~ y psittrrci rlio 6BC. Can. J . Microbiol.21: 759-763. Cl~lnmydio psittrzci 6BC attenue par culture sur cellules L e s t un antigene particulaire qui est phagocyte en grande quantite par les mononucleaires peritoneaux au cours de la Iere h qui suit I'inoculation intraperitoneale. Par la suite les phagocytes dttruisent les proprietts d'imrnunofluorescence des antigenes chlarnydiaux. On croit que la disparition rapide des antigenes I F des liquides peritoneaux est attribuable B une action rapide et dommageable des phagocytes, les chlarnydies induisant une activation d'enzyrnes lysosomales. [Traduit par le journal]

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Introduction The immune mechanisms functioning in chlamydial infections are largely unknown. Specific immunity to these parasites after recovery is not absolute (6,7,9). The reason why good immunological memory in chlamydioses is not induced is uncertain. Some authors (3) claim that this may be connected with the deleterious action of chlamydiae on macrophages and impairment of the processing of antigens. We have reported in a previous paper (5) that an L-cell-attenuated 6BC strain was cytopathic for peritoneal macrophages after inoculation into the mouse, not because of its ability to multiply in the mononuclear phagocytes, but because of the parasite's ability to damage the peritoneal phagocytes rapidly. The present studies are concerned with the fate of the immunofluorescent (IF) chlamydial antigens in peritoneal phagocytes after inoculation of L-cellattenuated 6BC strain into mice. Materials and Methods The studies reported here represent serial examinations performed in parallel with those described in the pre'Received August 29, 1974.

ceding paper (5). Mice were inoculated with Chlanzydia psittaci 6BC attenuated by growth in L cells (4). Peritoneal washings were collected l h and then daily up to 6 days after intraperitoneal inoculation of mice. Coverslip preparations of the cells from infected and non-infected controls were examined for the presence of chlamydial antigen by an indirect IF method. Ar~tiserum ngairrst C. psittaci 6BC was prepared as described before (4). The antiserum used in the work reported here was obtained from animals immunized with a heat-inactivated egg-attenuated 6BC strain. This antiserum showed no immunofluorescence using the indirect technique in cell preparations of mice inoculated with normaI, noninfected, L-cell homogenates. Other methods are described along with Results.

Results Our previous studies (2) on the fluorescence of chlamydial antigen in the peritoneal cells of mice infected with an egg-attenuated 6BC strain which multiplied in the mononuclear phagocytes have demonstrated considerable variation in the morphology, distribution, and localization of chlamydial antigens in these cells. Small lymphocytes "coated" with a thin layer of diffuse fluorescence appeared to be firmly attached to macrophages which exhibited specific staining. A marked increase of chlamydial antigen was Dresent in mononuclear cells in vivo as well as in cells that were maintained thereafter in vitro.

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CAN. J . MICROBIOL. VOL. 21, 1975

Epithelioid cells were observed in long-term cultures and demonstrated strong specific fluorescence. When peritoneal cells were harvested 1 h after inoculation of the L-cell-grown 6BC strain and the "settled" cell preparations examined by May-Griinwald-Giemsa staining, the search for chlamydial forms was difficult because of the poor spreading of the mononuclear phagocytes and the increased granulation of their cytoplasm. Peritoneal cells harvested 1 h after inoculation of mice with L-cell-attenuated 6BC and examined by I F revealed a large amount of specifically stained chlamydial antigen with a distribution, morphology, and localization (Figs. 1-5) similar to that observed a t 1, and up t o 48 h after infection of mice with egg-attenuated 6BC (4). The most significant difference in mononuclear phagocytes from mice inoculated with the two strains was seen from the 3rd day and up to 6 days after injection of mice, i.e. at a time when the egg-grown 6BC strain showed a marked increase of I F chlamydial forms in mononuclear phagocytes. In contrast, almost no I F antigen could be detected in peritoneal mononuclear phagocytes 3 to 6 days after injection of L-cellgrown 6BC (compare Fig. 1 1 with Figs. 9 and 12). Peritoneal cells harvested at varying intervals after infection in vivo and maintained thereafter in vitro were not suitable for detailed I F studies because of an early damage of these cells (Figs. 8 and 10). In mononuclear cells that spread poorly on cover slips but remained attached to the cover slips, the particulate antigen was associated with the surface membranes (Fig. 7

at 24 h and Fig. 8 at 4 days). In other macrophages which were apparently damaged, the particulate antigen had apparently been released (Fig. 10). Specific fluorescence has been detected only rarely in polymorphonuclear phagocytes (PMN's) and only shortly after inoculation (Fig. 6) probably because of the early cell damage. On the basis of these results and in accordance with our previous studies (5) it might be concluded that a large amount of particulate antigen of the L-cell-grown 6BC was phagocytized by peritoneal cells in vivo during the 1st h after infection, but that the lysosomal enzymes subsequently destroyed the I F properties of the parasite. The experiments have shown a clear difference between the fate of I F antigens of L-cell-grown vs. egg-grown 6BC strains in peritoneal phagocytes in vivo.

Discussion Many factors influence the persistence or degradation of microbial antigens in the intact host; macrophages are involved in the uptake and degradation of antigenic macromolecules. Bacteria and bacterial material enter reticuloendothelial cells in phagosomes and subsequently appear in phagolysosomes, where they are processed. Materials from lysosome-rich fractions of such cells have subsequently been found to be immunogenic. It is less clear that this sort of uptake and processing is a necessary step toward the acquisition of immunogenicity (I 1). It has been shown (10) that PMN's were more efficient in destroying Salmonella antigens than were peritoneal macrophages, although the latter was effective. Also, Cohn (2) reported that -

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ABBREVIATIONS USED: M, macrophage; N, polymorphonuclear leucocyte. Magnifications: Figs. 7-9, oil emersion (O.E.) x 400 ( x 4). Figs. 1-6 and 10-12. O.E. x 1000 ( x 6). FIG. 1. Cells harvested I h after inoculation of mice with homogenates of control, noninfected L-cellmaterial; with the exception of Figs. 9 and 12 (infected with egg-grown 6BC) all other micrographs are from mice inoculated with L-cell-grown 6BC. FIGS.2-5. Cells I h after infection; arrows point toward small empty cytoplasmic vesicles whose membranes show bright specific fluorescence. FIG. 6. Cells 1 day after inoculation ; N and small mononuclear cells adhering t o a large M. FIG.7. Cells 1 h after inoculation in vivo and maintained for 24 h in vitro; note poor spreading of mononuclear cells and particulate antigen predominantly on surface membranes. FIG. 8. Cells as in Fig. 7, but maintained for 4 days in vitro; note sparsity of cells and persistence of particulate antigen on the surface of poorly spread cells. FIG.9. Cells harvested 1 day after infection of mice with egg-grown 6BC and maintained for 4 days in vitro; note enlarged M with typical bright fluorescent chlamydial cytoplasmic inclusion. FIG. 10. Cells 1 day after inoculation of L-cell-grown 6BC maintained 24 h in vitro; note release of particulate antigen (arrows) from damaged cells. FIG. 11. Cells 5 days after inoculation as above; note faint fluorescence around empty cytoplasmic vesicles. FIG. 12. Cells harvested at the same period of time as above, but after infection of egg-grown 6BC: note marked increase of particulate antigen in well-defined cytoplasmic inclusion (arrows).

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KORDOVA E T AL.: CHLAMYDIA PSITTACI 6BC

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CAN. J. MICROBIOL. VOL. 21, 1975

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KORDOVA E T AL.: CHLAMYDIA PSITTACI 6BC

when E. coli was cultured in vitro for 2 h with rabbit peritoneal mononuclear phagocytes the immunogens were not destroyed, although the bacteria were disintegrated. On the other hand, PMN's readily destroyed these immunogens. I t has been suggested that the avidity with which PMN's ingest bacteria may indicate that PMN's have a more efficient degradative system (8). Damage of phagocytes may also contribute to enhanced dilution of antigens since damaged cells are themselves scavenged by other phagocytes (1). This may be worth emphasizing, since it may provide an explanation for the loss of IF-determinant groups in peritoneal cells of mice inoculated with the virulent (for mice) 6BC attenuated by growth in L cells. We are ignorant about the factors determining what makes some chlamydiae more "appetizing" than others for the "professional phagocytes" and there is obviously plenty of work required to define these factors.

Acknowledgments This research was supported by the Medical Research Council Grant No. MA 3901. We thank Mrs. Caroline Martin, Lynn Burton, and Shirley Wall for excellent assistance. 1. ASKONAS. B. A., and L. JAROSKOVA. 1970. Antigen in macrophages and antibody induction. In Mononuclear phagocytes. Edited by R. van Furth. Blackwell Scientific Publications, Oxford and Edinburgh. pp. 595-610.

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2. COHN,2. A. 1962. Influence of rabbit polymorphonuclear leukocytes and macrophages on the immunogenicity of Escherichia coli. Nature (Lond.), 196: 1066. 1971. 3. HOGG,A. E., L. M. C O L L I E R and , R. G. HARRIS. The antibody response to primary and repeated intravenous injection of TRIC agent. 111 Trachoma and related disorders. Eeliteel l7y R. L. Nichols. Excerpta Medica, Amsterdam, London. and Princeton. pp. 185-195. 4. KORDOVA, N., C. MARTIN,J. C. WILT, and W. MEYER. 1973. Immunofluorescence of peritoneal phagocytes after infection in vivo with egg-attenuated Cl1lar11?.c/icrpsittoci 6BC. Can. J. Microbiol. 19: 1425-1429. 5. KORDOVA, N., J . C. WILT, and C. MARTIN.1975. Lysosomes and the "toxicity" of Rickettsias. VI. In vivo response of mouse peritoneal phagocytes to psittcrci 6BC strain. Can. J. L-cell-grown Cltlrrt~~yclic~ Microbiol. 21: 323-33 I. 6. MEYER,K. F. 1965. Psittacosis-lymphogranuloma venereum agents. 111 Viral and rickettsia] infection of man. 4th ed. Edited by F. L. Horsfall and J. Tamm. Lippincott Co.. Philadelphia, Toronto. pp. 10061042. 1971. Trachoma and related 7. NICHOLS, R. L. (Eclitlito~.). disorders caused by chlamydia1 agents. Excerpta Medica, Princeton. pp. 560-568. 8. PERKINS,E . M. 1970. Digestion of antigen by peritoneal macrophages. In Mononuclear phagocytes. Eclitecll~.~ R. van Furth. Blackwell Scientific Publications, Oxford and Edinburgh. pp. 562-578. 9. STORZ,J. 1971. Chlamydia and Chlamydia-induced diseases. C. C. Thomas, Springfield, Illinois. 10. WALSH,T . E.. and C. A. S M I T H1951. . The influence of polymorphonuclear leukocytes and macrophages on antibody production. J. Immunol. 66: 303-3 10. 11. W E I S S M A N N G., 1967. The role of lysosomes in inflammation and disease. Annu. Rev. Med. 18: 97-1 12.

Immunofluorescence of peritoneal phagocytes after infection of mice with L-cell-attenuated Chlamydia psittaci 6BC.

Large amounts of particulate antigen of Chlamydia psittaci 6BC attenuated by growth in L cells were phagocytized by peritoneal mononuclear phagocytes ...
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