INFECTION AND IMMUNITY, Aug. 1976, p. 416-421 Copyright © 1976 American Society for Microbiology
Vol. 14, No. 2
Printed in U.S.A.
Interaction Between Coxiella burnetii and Guinea Pig Peritoneal Macrophages R. A. KISHIMOTO* AND J. S. WALKER' United States Army Medical Research Institute of Infectious Diseases, Frederick, Maryland 21701 Received for publication 12 March 1976
The phagocytosis and subsequent degradation of phase I and II Coxiella burnetii by macrophages obtained from immune and nonimmune guinea pigs were compared. Phase I rickettsiae were more resistant to phagocytosis than were phase II organisms. There was no significant difference in the percentage of phagocytosis of either phase of rickettsiae by macrophages from immune or nonimmune animals. After ingestion, phase I and II organisms pretreated with normal serum multiplied and destroyed normal macrophages as well as macrophages obtained from guinea pigs immunized with phase II rickettsiae. In contrast, only phase I organisms were degraded by macrophages from phase Iimmunized animals in the presence of normal serum. Immune serum rendered rickettsiae more susceptible to phagocytosis and also potentiated the destruction of organisms by all types of macrophages. The specificity of macrophages from phase I animals to degrade only phase I rickettsiae was demonstrated by the ability of Rickettsia rickettsii to replicate in these macrophages. The pathogenesis of Q fever and mechanisms of immunity against this disease are not well known. Among reports are those of Brezina and Kazar (3) and Wisseman et al. (24), who found that polymorphonuclear leukocytes are capable of phagocytizing Coxiella burnetii. Macrophages have been implicated in pathogenesis, but the mechanism by which they perform their function has not been fully explained (23). Conflicting results on the interaction between phase I and II C. burnetii with macrophages have been reported. Downs (5) found that phase II rickettsiae were more resistant to phagocytosis by normal macrophages than were phase I organisms, whereas Kazar et al. (12) concluded the reverse to be true. The fate of ingested rickettsiae was not resolved by Downs or Kazar et al. The disparate results obtained by these investigators indicate gaps in the knowledge of certain aspects of immunity against C. burnetii, which suggests a need for additional study. The present study was undertaken to determine whether immunological specificity to Q fever is associated with macrophages. We have conducted experiments to determine the ability of macrophages from immune and nonimmune guinea pigs to phagocytize and kill ingested rickettsiae and whether these reactions are immunologically specific. MATERIALS AND METHODS Preparation of rickettsial stock suspensions. The Present address: Plum Island Animal Disease Center, P.O. Box 949, Greenport, Long Island, NY 11944.
third egg passage of Henzerling strain of C. burnetii in phase I was obtained from the Merrell-National Drug Co., Philadelphia, Pa. The 88th egg passage of the phase II Nine-Mile strain was from a stock culture of our laboratories. Chicken embryo cells were prepared and grown in roller bottles according to the method of Kenyon and Pedersen (13). The monolayer cell culture was infected with 15 ml of a 10% rickettsial stock suspension (2 x 106/ml) in Hanks balanced salt solution (HBSS). During the infection period the bottles were revolved at 0.4 rpm for 1 h at 35°C; the residual inoculum was removed, and 200 ml of Earle medium 199 (GIBCO, Grand Island, N.Y.) containing 2% fetal calf serum was added. The cultures were incubated on the roller apparatus (0.4 rpm) for 9 to 10 days at 35°C. Infected cell cultures were alternately frozen and thawed to release intracellular rickettsiae. Cellular debris was permitted to settle; the rickettsiae in the supernatant fluid were pelleted by centrifugation at 8,400 x g for 2 h. Rickettsiae were resuspended in Earle medium 199 supplemented with 2% fetal calf serum and stored at - 70°C. Rickettsial counts were determined by the method of Silberman and Fiset (19). The median infectious dose of the rickettsial suspension was estimated by intraperitoneal titration in outbred white mice [Tac:(SW)fBR]. Sera. Outbred Hartley strain guinea pigs, ing 350 to 450 g, obtained from Buckberg LabweighAnimals, Tompkin Cove, N.Y., were immunized by intraperitoneal injection at 4-week intervals with 0.2 ml of organism suspension containing approximately 109 formalin-killed phase I or II C. burnetii. Serum complement-fixing antibody was determined 2 weeks after the last immunization. Serum complement-fixing titers from phase I-immunized guinea pigs were 1:128 to 1:256 against homologous antigen and 1:32 to 1:64 against heterologous antigen. Phase
416
VOL. 14, 1976
C. BURNETII-MACROPHAGE INTERACTION
417
II-immunized guinea pigs usually had a titer of 1:256 the rickettsiae in 100 macrophages. Because of the to 1:512 against phase II antigen and 1:16 to 1:32 difficulty in counting intracellular rickettsiae, a against heterologous antigen. Sera obtained from scoring system of infected cells similar to that of nonimmunized guinea pigs had no detectable com- Gambrill and Wisseman (7) was employed. Individplement-fixing antibody titers. ual rickettsiae were counted in cells containing 1 to Preparation of peritoneal macrophages. Perito- 10 organisms. Those cells containing 11 to 24 organeal macrophages were harvested 10 to 12 days after nisms were scored as 17, 25 to 49 were scored as 37, the last injection. Macrophages were collected 4 and those having more than 50 were scored as 50. days after intraperitoneal injection of guinea pigs Inability to observe stained rickettsiae at later time with 25 ml of mineral oil (Marcol no. 90; Humble Oil periods, after they were initially observed at 60 min, & Refining Co., Houston, Tex.). The peritoneal exu- was interpreted as evidence of rickettsial degradadate cells were harvested and processed according to tion. Occasionally, a cell that resembled a large the method of David et al. (4). Approximately 106 lymphocyte was observed in stained preparations. macrophages in 1 ml were dispensed into Leighton The incidence of these cells never exceeded 1% of the tubes containing cover slips and incubated at 37°C total cell population. for 2 h. Nonadherent cells were removed by three 5Statistical analysis. The chi-square test with ml washes with HBSS, 1 ml of fresh Earle medium Yates' correction was used to determine statistical 199 supplemented with 10% fetal calf serum was significance of the difference of the percentage of added, and the cells were incubated an additional 16 phagocytosis of phase I and II rickettsiae by macroto 18 h. phages in the presence of normal or immune serum Exposure of macrophages to rickettsiae. The ex- and the average number of organisms per infected perimental design for the selected macrophage-C. cell. The level of significance was set at P < 0.05. burnetii-serum interaction is shown in Table 1. Approximately 108 phase I or II C. burnetii in 1.0 ml RESULTS was incubated in either normal or homologous immune heat-inactivated serum for 30 min at 37°C, Phagocytosis of C. burnetii. The interaction then added to the macrophages, and incubated an additional 60 min. The inoculum was removed by between phase I and II rickettsiae with macroaspiration, and the macrophage culture was washed phages from immune and nonimmune guinea three times with 5 ml of HBSS. Cultures to be held pigs in selected sera is summarized in Table 2. for longer periods were given fresh Earle medium Phase I rickettsiae treated with normal serum 199 containing 10% fetal calf serum and incubated at were significantly more resistant to phagocyto37°C. Infected macrophages were washed daily two sis by the different types of macrophages than times with HBSS, and fresh medium was added. No were phase II organisms (P < 0.001). Also, the antibiotics were used in this study. Quantitation of intracellular rickettsiae. Cover average number of phase I rickettsiae per inslips from Leighton tube preparations were fixed in fected cell was less than that of phase II orgaabsolute methanol for 10 min, air dried, and stained nisms (P < 0.001). When phase I and II orgaby a modified method of Gimenez (8). Cells were nisms were treated with homologous antiserum immersed in buffered basic carbol fuchsin for 4 min, prior to interaction, significantly more macrowashed in distilled water for 30 s, and then counter- phages from immune and nonimmune animals stained with malachite green for 30 s. Stained cover contained rickettsiae (P < 0.05). Additionally, slips were mounted in Permount (Fisher Scientific the average number of organisms per infected Co., Pittsburgh, Pa.) and examined with an oil im- cell also increased (P < 0.005), with the excepmersion objective. Duplicate samples were exam- tion of phase I rickettsiae in normal macroined at 60 min (base line) and 1, 2, 3, and 5 days after infection. The percentage of macrophages which phages. The effects of strain differences were contained one or more rickettsiae within their cell also investigated. When the third egg passage boundaries as well as the average number of rickett- of a Nine-Mile strain in phase I was substituted siae per infected cell was determined by counting for the phase I Henzerling strain, the phagoTABLE 1. Experimental design: Expt design 1. Macrophage Serum Phase of C. burnetii
selected combinations of macrophage-C. burnetii-serum interaction Normal Normal I or II
2. Macrophage Serum Phase of C. burnetii
Phase I immune Normal I or II
3. Macrophage Serum Phase of C. burnetii
Phase II immune Normal I or II
Variables Normal Anti-phase I I
Normal
Anti-phase II II
Phase I immune
Phase I immune
Anti-phase I
Anti-phase II
I
II
Phase II immune
Phase II immune
Anti-phase I
Anti-phase II
I
II
418
INFECT. IMMUN.
KISHIMOTO AND WALKER
TABLE 2. Phagocytosis of C. burnetii by different types of macrophages in selected sera Avg no. of rickettsiae/infected
Phagocytosis (%)
Type of macrophage
cell
Type of serum Phase I
Phase II
7 Normal 80 Immune 98b 69a Phase I Normal 12 82 immuned Immune 93a 92b Phase II Normal 10 84 immuned Immune 89a 97a a p < 0.001, immune versus normal. bp < 0.05, immune versus normal. Immune versus normal reading was not significant. d Comparisons of phase I to phase II in normal serum are significant, P eP < 0.02, immune versus normal.
Phase I
Phase II
4
20 41a 27 40e 28 43b
Normal
cytic uptake and subsequent fate of ingested organisms were identical. Fate of ingested rickettsiae. The fate of ingested rickettsiae in macrophages is summarized in Fig. 1. The average number of phase I and II C. burnetii previously treated with normal serum increased within normal macrophages after phagocytosis. The number of phase I and II organisms increased more than 12-fold and 2-fold, respectively, in 5 days (Fig. 1A). Attempts were made to quantitate replication or killing of rickettsiae within macrophages. At selected times after infection macrophages were frozen and thawed three times, and the lysate was diluted in HBSS and then cultured in chicken-embryonated eggs, WI-38 cells, and chicken fibroblast cells. However, the results were not consistent. This was probably due to the insensitivity of the test. However, our subsequent transmission electron microscopic observations on the interaction between C. burnetii and normal macrophages have confirmed that rickettsiae multiplied freely in macrophage phagosomes (Kishimoto et al., submitted for publication). However, phase I organisms could not be detected after 5 days in macrophages from phase I-immunized guinea pigs (Fig. 1B). In the heterologous system, growth of phase I rickettsiae was inhibited for 1 to 3 days within macrophages obtained from phase II-immunized guinea pigs, but replication occurred 3 to 5 days after infection (Fig. 1C). Phase I rickettsiae treated with homologous antiserum prior to interaction was degraded by all types of macrophages within 3 to 5 days (Fig. lA-C). Phase II rickettsiae previously treated with normal serum multiplied freely within normal macrophages after ingestion. Infected macrophages appeared intact and viable for 1 to 2 days (by trypan blue exclusion). However, 3 to 5 days later the few remaining adherent macro-
7'
3 34a 5 20a
Vol. 14, No. 2
Printed in U.S.A.
Interaction Between Coxiella burnetii and Guinea Pig Peritoneal Macrophages R. A. KISHIMOTO* AND J. S. WALKER' United States Army Medical Research Institute of Infectious Diseases, Frederick, Maryland 21701 Received for publication 12 March 1976
The phagocytosis and subsequent degradation of phase I and II Coxiella burnetii by macrophages obtained from immune and nonimmune guinea pigs were compared. Phase I rickettsiae were more resistant to phagocytosis than were phase II organisms. There was no significant difference in the percentage of phagocytosis of either phase of rickettsiae by macrophages from immune or nonimmune animals. After ingestion, phase I and II organisms pretreated with normal serum multiplied and destroyed normal macrophages as well as macrophages obtained from guinea pigs immunized with phase II rickettsiae. In contrast, only phase I organisms were degraded by macrophages from phase Iimmunized animals in the presence of normal serum. Immune serum rendered rickettsiae more susceptible to phagocytosis and also potentiated the destruction of organisms by all types of macrophages. The specificity of macrophages from phase I animals to degrade only phase I rickettsiae was demonstrated by the ability of Rickettsia rickettsii to replicate in these macrophages. The pathogenesis of Q fever and mechanisms of immunity against this disease are not well known. Among reports are those of Brezina and Kazar (3) and Wisseman et al. (24), who found that polymorphonuclear leukocytes are capable of phagocytizing Coxiella burnetii. Macrophages have been implicated in pathogenesis, but the mechanism by which they perform their function has not been fully explained (23). Conflicting results on the interaction between phase I and II C. burnetii with macrophages have been reported. Downs (5) found that phase II rickettsiae were more resistant to phagocytosis by normal macrophages than were phase I organisms, whereas Kazar et al. (12) concluded the reverse to be true. The fate of ingested rickettsiae was not resolved by Downs or Kazar et al. The disparate results obtained by these investigators indicate gaps in the knowledge of certain aspects of immunity against C. burnetii, which suggests a need for additional study. The present study was undertaken to determine whether immunological specificity to Q fever is associated with macrophages. We have conducted experiments to determine the ability of macrophages from immune and nonimmune guinea pigs to phagocytize and kill ingested rickettsiae and whether these reactions are immunologically specific. MATERIALS AND METHODS Preparation of rickettsial stock suspensions. The Present address: Plum Island Animal Disease Center, P.O. Box 949, Greenport, Long Island, NY 11944.
third egg passage of Henzerling strain of C. burnetii in phase I was obtained from the Merrell-National Drug Co., Philadelphia, Pa. The 88th egg passage of the phase II Nine-Mile strain was from a stock culture of our laboratories. Chicken embryo cells were prepared and grown in roller bottles according to the method of Kenyon and Pedersen (13). The monolayer cell culture was infected with 15 ml of a 10% rickettsial stock suspension (2 x 106/ml) in Hanks balanced salt solution (HBSS). During the infection period the bottles were revolved at 0.4 rpm for 1 h at 35°C; the residual inoculum was removed, and 200 ml of Earle medium 199 (GIBCO, Grand Island, N.Y.) containing 2% fetal calf serum was added. The cultures were incubated on the roller apparatus (0.4 rpm) for 9 to 10 days at 35°C. Infected cell cultures were alternately frozen and thawed to release intracellular rickettsiae. Cellular debris was permitted to settle; the rickettsiae in the supernatant fluid were pelleted by centrifugation at 8,400 x g for 2 h. Rickettsiae were resuspended in Earle medium 199 supplemented with 2% fetal calf serum and stored at - 70°C. Rickettsial counts were determined by the method of Silberman and Fiset (19). The median infectious dose of the rickettsial suspension was estimated by intraperitoneal titration in outbred white mice [Tac:(SW)fBR]. Sera. Outbred Hartley strain guinea pigs, ing 350 to 450 g, obtained from Buckberg LabweighAnimals, Tompkin Cove, N.Y., were immunized by intraperitoneal injection at 4-week intervals with 0.2 ml of organism suspension containing approximately 109 formalin-killed phase I or II C. burnetii. Serum complement-fixing antibody was determined 2 weeks after the last immunization. Serum complement-fixing titers from phase I-immunized guinea pigs were 1:128 to 1:256 against homologous antigen and 1:32 to 1:64 against heterologous antigen. Phase
416
VOL. 14, 1976
C. BURNETII-MACROPHAGE INTERACTION
417
II-immunized guinea pigs usually had a titer of 1:256 the rickettsiae in 100 macrophages. Because of the to 1:512 against phase II antigen and 1:16 to 1:32 difficulty in counting intracellular rickettsiae, a against heterologous antigen. Sera obtained from scoring system of infected cells similar to that of nonimmunized guinea pigs had no detectable com- Gambrill and Wisseman (7) was employed. Individplement-fixing antibody titers. ual rickettsiae were counted in cells containing 1 to Preparation of peritoneal macrophages. Perito- 10 organisms. Those cells containing 11 to 24 organeal macrophages were harvested 10 to 12 days after nisms were scored as 17, 25 to 49 were scored as 37, the last injection. Macrophages were collected 4 and those having more than 50 were scored as 50. days after intraperitoneal injection of guinea pigs Inability to observe stained rickettsiae at later time with 25 ml of mineral oil (Marcol no. 90; Humble Oil periods, after they were initially observed at 60 min, & Refining Co., Houston, Tex.). The peritoneal exu- was interpreted as evidence of rickettsial degradadate cells were harvested and processed according to tion. Occasionally, a cell that resembled a large the method of David et al. (4). Approximately 106 lymphocyte was observed in stained preparations. macrophages in 1 ml were dispensed into Leighton The incidence of these cells never exceeded 1% of the tubes containing cover slips and incubated at 37°C total cell population. for 2 h. Nonadherent cells were removed by three 5Statistical analysis. The chi-square test with ml washes with HBSS, 1 ml of fresh Earle medium Yates' correction was used to determine statistical 199 supplemented with 10% fetal calf serum was significance of the difference of the percentage of added, and the cells were incubated an additional 16 phagocytosis of phase I and II rickettsiae by macroto 18 h. phages in the presence of normal or immune serum Exposure of macrophages to rickettsiae. The ex- and the average number of organisms per infected perimental design for the selected macrophage-C. cell. The level of significance was set at P < 0.05. burnetii-serum interaction is shown in Table 1. Approximately 108 phase I or II C. burnetii in 1.0 ml RESULTS was incubated in either normal or homologous immune heat-inactivated serum for 30 min at 37°C, Phagocytosis of C. burnetii. The interaction then added to the macrophages, and incubated an additional 60 min. The inoculum was removed by between phase I and II rickettsiae with macroaspiration, and the macrophage culture was washed phages from immune and nonimmune guinea three times with 5 ml of HBSS. Cultures to be held pigs in selected sera is summarized in Table 2. for longer periods were given fresh Earle medium Phase I rickettsiae treated with normal serum 199 containing 10% fetal calf serum and incubated at were significantly more resistant to phagocyto37°C. Infected macrophages were washed daily two sis by the different types of macrophages than times with HBSS, and fresh medium was added. No were phase II organisms (P < 0.001). Also, the antibiotics were used in this study. Quantitation of intracellular rickettsiae. Cover average number of phase I rickettsiae per inslips from Leighton tube preparations were fixed in fected cell was less than that of phase II orgaabsolute methanol for 10 min, air dried, and stained nisms (P < 0.001). When phase I and II orgaby a modified method of Gimenez (8). Cells were nisms were treated with homologous antiserum immersed in buffered basic carbol fuchsin for 4 min, prior to interaction, significantly more macrowashed in distilled water for 30 s, and then counter- phages from immune and nonimmune animals stained with malachite green for 30 s. Stained cover contained rickettsiae (P < 0.05). Additionally, slips were mounted in Permount (Fisher Scientific the average number of organisms per infected Co., Pittsburgh, Pa.) and examined with an oil im- cell also increased (P < 0.005), with the excepmersion objective. Duplicate samples were exam- tion of phase I rickettsiae in normal macroined at 60 min (base line) and 1, 2, 3, and 5 days after infection. The percentage of macrophages which phages. The effects of strain differences were contained one or more rickettsiae within their cell also investigated. When the third egg passage boundaries as well as the average number of rickett- of a Nine-Mile strain in phase I was substituted siae per infected cell was determined by counting for the phase I Henzerling strain, the phagoTABLE 1. Experimental design: Expt design 1. Macrophage Serum Phase of C. burnetii
selected combinations of macrophage-C. burnetii-serum interaction Normal Normal I or II
2. Macrophage Serum Phase of C. burnetii
Phase I immune Normal I or II
3. Macrophage Serum Phase of C. burnetii
Phase II immune Normal I or II
Variables Normal Anti-phase I I
Normal
Anti-phase II II
Phase I immune
Phase I immune
Anti-phase I
Anti-phase II
I
II
Phase II immune
Phase II immune
Anti-phase I
Anti-phase II
I
II
418
INFECT. IMMUN.
KISHIMOTO AND WALKER
TABLE 2. Phagocytosis of C. burnetii by different types of macrophages in selected sera Avg no. of rickettsiae/infected
Phagocytosis (%)
Type of macrophage
cell
Type of serum Phase I
Phase II
7 Normal 80 Immune 98b 69a Phase I Normal 12 82 immuned Immune 93a 92b Phase II Normal 10 84 immuned Immune 89a 97a a p < 0.001, immune versus normal. bp < 0.05, immune versus normal. Immune versus normal reading was not significant. d Comparisons of phase I to phase II in normal serum are significant, P eP < 0.02, immune versus normal.
Phase I
Phase II
4
20 41a 27 40e 28 43b
Normal
cytic uptake and subsequent fate of ingested organisms were identical. Fate of ingested rickettsiae. The fate of ingested rickettsiae in macrophages is summarized in Fig. 1. The average number of phase I and II C. burnetii previously treated with normal serum increased within normal macrophages after phagocytosis. The number of phase I and II organisms increased more than 12-fold and 2-fold, respectively, in 5 days (Fig. 1A). Attempts were made to quantitate replication or killing of rickettsiae within macrophages. At selected times after infection macrophages were frozen and thawed three times, and the lysate was diluted in HBSS and then cultured in chicken-embryonated eggs, WI-38 cells, and chicken fibroblast cells. However, the results were not consistent. This was probably due to the insensitivity of the test. However, our subsequent transmission electron microscopic observations on the interaction between C. burnetii and normal macrophages have confirmed that rickettsiae multiplied freely in macrophage phagosomes (Kishimoto et al., submitted for publication). However, phase I organisms could not be detected after 5 days in macrophages from phase I-immunized guinea pigs (Fig. 1B). In the heterologous system, growth of phase I rickettsiae was inhibited for 1 to 3 days within macrophages obtained from phase II-immunized guinea pigs, but replication occurred 3 to 5 days after infection (Fig. 1C). Phase I rickettsiae treated with homologous antiserum prior to interaction was degraded by all types of macrophages within 3 to 5 days (Fig. lA-C). Phase II rickettsiae previously treated with normal serum multiplied freely within normal macrophages after ingestion. Infected macrophages appeared intact and viable for 1 to 2 days (by trypan blue exclusion). However, 3 to 5 days later the few remaining adherent macro-
7'
3 34a 5 20a
