69
Macrophage-Mediated Killing of Opsonized Treponema pallidum Sharon A. Baker-Zander and Sheila A. Lukehart
Department of Medicine, University of Washington School ofMedicine, Seattle
Macrophages are thought to be responsible for the rapid clearance of Treponema pal/idum subspecies pal/idum from early syphilitic lesions. While studies in vivo and in vitro strongly support this hypothesis, the ability of macrophages to kill T. pal/idum has not been demonstrated. In natural and experimental syphilis infection, lymphocyte and macrophage infiltration in lesions [1-3] and increased numbers of monocytes in peripheral blood [4-7] and macrophages in other lymphoid organs [8] have been well characterized. Light and electron microscopic evaluation oflesion biopsies detect both intact and degenerating treponemes in vacuoles within macrophages, suggesting active phagocytosis and dissolution of T. pal/idum [1, 2, 9, 10]. We have previously demonstrated T. pallidum antigens within macrophages in healing primary lesions in the rabbit model [9,11,12]. Lukehart and Miller [13] documented active phagocytosis of viable T. pallidum by proteose peptone-induced normal rabbit peritoneal macrophages in vitro; organisms were identified within vacuoles by immunofluorescence staining and electron microscopy. Phagocytosis was significantly enhanced by opsonization with immune serum, although a role for complement was not demonstrated. More recently, Alder et a1. [14] confirmed treponemal phagocytosis using hamster peritoneal macrophages and a different subspecies of T. pallidum attached to polycarbonate filters. Again, phagocytosis was increased with immune serum and proceeded slowly. In neither study, however, was the actual killing of organisms by macrophages examined. Some evidence for cell-mediated extracellular treponemiReceived I July 1991; revised 23 September 1991. Grant support: National Institutes of Health (AI-18988). Reprints or correspondence: Sharon A. Baker-Zander, Department of Medicine/Infectious Diseases, ZA-20, Harborview Medical Center, 325 Ninth Ave., Seattle, WA 98104. The Journal ofInfectious Diseases 1992;165:69-74 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6501-0008$01.00
cidal mechanisms also exists. Spleen cell preparations cultured in the presence oftreponemal antigen produce soluble factors that reportedly immobilize and kill T. pallidum [15, 16]. The present report documents killing of T. pal/idum after ingestion by macrophages in vitro.
Materials and Methods Animals. Adult male New Zealand white rabbits were obtained from R & R Rabbitry (Stanwood, WA) and were housed individually at 18-20°C and given antibiotic-free food and water. Rabbits were examined on receipt for serologic evidence of infection with Treponema paraluiscuniculi by the VDRL and fluorescent treponemal antibody-absorbed (FTA-ABS) tests. These tests were done as recommended by the Centers for Disease Control [17] with modifications for use with rabbit sera [11]. Organisms. T. pallidum pal/idum (Nichols strain) was maintained by intratesticular passage in rabbits as previously described [18]. For experiments, organisms were extracted from infected testes in sterile medium 199 (M 199). Gross testicular debris was sedimented by low-speed centrifugation (350 g for 7 min), and organisms in the supernatant were enumerated by darkfield microscopy and diluted in M 199 to 2 X 107/rnl for immunofluorescence studies or 2 X 105 /ml for the in vitro killing assay. Sera. Pools of normal rabbit sera (NRS, VDRL and FTAABS nonreactive) and immune rabbit sera (IRS, from rabbits infected with T. pal/idum either intratesticularly or intradermally for 2-10 months) were prepared. Blood was collected from the medial ear artery into sterile glass tubes and allowed to clot at 4°C overnight. Tubes were spun at 750 g, and the serum was collected, pooled, and filter-sterilized. Pools were then heatinactivated at 56°C for 30 min, dispensed in 3-ml aliquots, and stored frozen at -20°C until use. Specific antitreponemal antibodies in the IRS pool were evaluated by titration in the FTAABS test and by immunoblot against treponemal antigens as described elsewhere [19]. Pooled human syphilitic serum (IHS) was prepared from
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The ability of proteose peptone-induced normal rabbit peritoneal macrophages to kill Treponema pallidum subspecies pallidum in vitro is demonstrated. Treponemes and 10% heated immune or normal sera were incubated with macrophages at a ratio of 1:200. After 2-10 h of incubation, these mixtures were injected intradermally at duplicate sites on normal rabbits. Maximal killing (failure to develop lesions) was seen at 10 h of incubation with immune serum: Only 7% (1/14) of lesions developed compared with 90% (9/10) after incubation in the presence of normal serum (P < .001). Maximal phagocytosis (detected by immunofluorescence) occurred by 8 h in the presence of immune serum, when 90% of macrophages had ingested treponemes. At this point, however, 70% of lesion sites from macrophages incubated with treponemes and immune serum still developed, suggesting that effective killing may require at least 2 h after phagocytosis.
70
Baker-Zander and Lukehart
were counted for each coverslip. The reader was blinded to the identity of each specimen. Data are expressed as mean percentage of cells with labeled vacuoles. Control cultures omitting T. pal/idum were routinely examined in parallel and averaged 1%2% positive. Killing assay. Killing after phagocytosis was evaluated by determining the absence or delay of lesion development after inoculation of T. pal/idum (both cell-associated and extracellular treponemes) after incubation for various times with macrophages or fibroblasts. Macrophage and fibroblast cultures (sixwell plates) were rinsed three times with warm M 199, and 1.9 ml of fresh M 199 supplemented with glutamine and containing either 20% NRS or 10% NRS and 10% IRS (but no antibiotics) was added to the cultures. Freshly extracted T. pal/idum ( 100 JLI ofa suspension of2 X 105 treponemes/rnl) were then added to each culture, yielding a final macrophage-to-treponeme ratio of 200: 1. Cultures were incubated anaerobically as described earlier at 37°C to promote treponemal survival. At various times up to 12 h, macrophages were scraped from the plate surface into the culture medium using a cell scraper, and macrophagetreponeme mixtures were inoculated intradermally at duplicate sites into the clipped backs of seronegative normal rabbits (100JLl vel/site, equal to 103 treponemes initially added in culture). Rabbits were observed for lesion development for 30 days; the absence or delay oflesion development was a direct reflection of decreased numbers of viable T. pal/idum. Five separate macrophage preparations, inoculated into a total of 19 rabbits, were examined. Data analysis. The means ± SE were calculated forimmunofluorescence data; comparisons between groups were done by analysis of variance. Fisher's exact test was used to compare the proportion of lesions that developed in each group. Statistical significance was defined as P < .05.
Results Evaluation of antitreponemal antibodies in NRS and IRS pools used for opsonization. Pooled IRS was evaluated in the FTA-ABS test using serial twofold dilutions of antibody in PBS after initial 1:5 dilution in sorbent. The highest dilution that still gave 2+ fluorescence was I: 1024. The NRS pool was nonreactive in the FTA-ABS test. Both sera were examined by immunoblot against SDSPAGE-separated T. pallidum antigens. The NRS pool diluted 1: 100 failed to detect any treponemal antigens, whereas the IRS pool detected a full complement oftreponemal antigens described previously [19], even at a dilution of 1:500. Phagocytosis of T. pal/idum by peritoneal macrophages. The degree of phagocytosis of viable T. pal/idum by proteose peptone-induced normal peritoneal macrophages in culture was determined by immunofluorescence staining using IHS and FITC-conjugated anti-human IgG. Ingestion in the presence of 20% NRS was compared with that in the presence of 10% NRS plus 10% IRS over a 10-h period (figure 1). Maximal phagocytosis was reached by 8 h of incubation in IRS,
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serum specimens collected from patients diagnosed with primary, secondary, or latent syphilis. Sera were heat-inactivated, dispensed, and stored as above. Rabbit fibroblasts. To provide a control for treponemal survival in the presence ofa nonphagocytic cell line, primary rabbit testicular fibroblast cultures were derived from normal testicular tissue by the method of Fitzgerald et al. [20]. Testes were minced and digested in 0.25% trypsin and 0.0 I% EDTA in PBS (pH 7.8) for 15 min on a magnetic stirrer. The resulting mixture was centrifuged at 400 g for 10 min, and the pellet was washed once in Eagle's MEM with Hanks' salts (MEM-HS) to remove the trypsin. The pellet was suspended in MEM-HS containing 20 mM HEPES, 10% fetal bovine serum, 1% 50X amino acids, 2% 100X vitamins, I%glutamine, and I%penicillin and streptomycin and dispensed into T-150 flasks (Costar, Cambridge, MA). Cultures were maintained at 37°C in 5% CO 2 and air. Medium was changed three times weekly, and fibroblasts were subcultured when confluent. One day before macrophage killing assays, confluent fibroblast cultures were trypsinized, washed twice in complete MEMHS, counted by hemocytometer, and adjusted to 105-106 cells/ ml. Fibroblasts were dispensed in six-well cluster plates (2 ml/well: Costar) and cultured overnight. Preparation ofmacrophages. Rabbit peritoneal macrophages were induced by intraperitoneal injection of 10 ml of 15%sterile proteose peptone 4 days before harvesting by peritoneal lavage with balanced salt solution (BSS) containing heparin (10 units/ ml). Cells were washed three times in cold BSS, counted by hemocytometer, and adjusted to 2 X 106/rnl in M 199 supplemented with 20% heat-inactivated NRS, penicillin, streptomycin, and glutamine. Macrophages were dispensed in 6- or 24well cluster tissue culture plates (2 or 0.5 rnl/well; Costar) and incubated for 2 h at 37°C in 5% CO 2 • Nonadherent cells were removed by mild agitation, fresh medium was added, and the cultures were incubated overnight. Before incubation with treponemes, cultures were washed to remove residual antibiotics. Phagocytosis. For immunofluorescent detection oftreponemal antigens after phagocytosis, macrophages were cultured in 24-well plates (0.5 mljwell) containing sterile glass coverslips in an anaerobic environment generated by anaerobic system envelopes (Difco, Detroit) in Gaspak jars (BBL Microbiology Systems, Cockeysville, MD). At various times after coculture with 107 freshly extracted treponemes, coverslips were rinsed with warm M 199 and fixed in 95% ethanol. Fixed cells on coverslips were rehydrated with PBS for 10 min at room temperature, then incubated with IHS (diluted 1:50 in PBS containing 1% bovine serum albumin) for 30 min at 37°C. After this incubation, coverslips were washed three times with PBS and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-human IgG (Zymed Laboratories, San Francisco) diluted I: 1000 in PBS containing 2% Tween 80 for 30 min at 37°C. Coverslips were again washed three times in PBS, allowed to air dry, and inverted on microscope slides with fluorescence mounting medium. Coverslips were examined under a Zeiss fluorescence microscope for the presence of fluorescein-labeled treponemal antigen in vacuoles within macrophages. Triplicate specimens were prepared for each time point or condition, and 100 macrophages
1ID 1992; 165 (January)
Macrophage Killing of T. pallidum
110 1992; 165 (January)
100-r---------------------. IRS 0)
c
~
80
CD
0)
c
-;;e:
CD:::J
O)~ «S:::::: .s::tU Q.Q"
eJ..,: o
60 NRS
40
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0
20
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0
Figure 1.
Rate of phagocytosis of Treponema pal/idum by normal rabbit peritoneal macrophages in the presence of normal (NRS) or immune rabbit serum (IRS). Triplicate cultures ofmacrophages were incubated with viable T. pal/idum for various times and fixed and stained by immunofluorescence for treponemal antigens. Data points represent mean ± SE percentage of macrophages containing fluorescent-stained treponemes. Differences between NRS and IRS were significant (P < .0 I) by 2 h.
when >90% of macrophages contained ingested fluorescent treponemal antigens. At this time, fewer than 45% of macrophages incubated with treponemes and NRS contained any treponemal antigens (P < .0 I, compared with IRS). Killing after in vitro phagocytosis of T. pallidum. The ability of rabbit peritoneal macrophages to successfully kill ingested T. pallidum is shown in table I. Maximal killing was seen after 10 h ofincubation; only I (7%) of 14 lesions developed from macrophages incubated with treponemes and 10% IRS, compared with 9 (90%) of 10 after incubation with NRS (P < .001). The single lesion that developed in the IRS group was delayed 4.4 days compared with the corresponding NRS
Table 1.
control (26 days for IRS vs. 21.6 ± 1.1 days for NRS). This indicates that only a small portion ofthe original treponemes in this one sample survived the 10-h incubation. The mean time to lesion development after 10 h of incubation with macrophages and NRS was comparable to that of treponemes and fibroblasts incubated in either NRS or IRS (20.7 and 19.0 days, respectively), suggesting that no macrophagemediated killing of T. pal/idum occurred in the presence ofNRS. In this bacterial killing assay, extracellular replication of T. pallidum is not a confounding issue, even though the incubation times were as long as 10 h, because the organism's maximum generation time is 33 h in vivo [21] and is estimated to be often slower in vitro [22, 23]. Virtually no killing was detected after only 4 h of incubation, but an increasing delay in lesion development and fewer numbers of lesions observed thereafter (compared with NRS) suggests that progressively more killing occurred as incubation times were increased. Effects ofculture conditions and incubation time on T. pallidum survival. To demonstrate that the loss ofinfectivity of treponemal suspensions was not due to in vitro incubation conditions alone, cultures ofrabbit testicular fibroblasts were incubated with T. pallidum under identical experimental conditions. Treponemal survival was measured as mean time to onset oflesion development after intradermal inoculation of rabbits (table 1). After a 10-h incubation in the presence of decomplemented NRS, lesions were observed at all sites (6/6) in a mean time of20.7 ± 0.3 days; injection ofcultures incubated in IRS yielded 5 (83.3%) of 6 lesions in a mean time of 19.0 ± 1.2 days. This delay of -- 6 days (beyond the expected onset of lesion development of 13 days if all 103 treponemes had survived) probably reflects a loss oftreponemal viability due to incubation conditions equivalent to 1 log [21]. No treponemicidal effects of incubation in IRS compared with NRS were observed with fibroblasts.
Demonstration of intracellular killing of T. pallidum. Days to lesion development (mean ± SE)
Hours*
Lesions/total sites (%)
NRS
IRS
NRS
IRS
pt
15.5 ± 1.9 15.7±0.9 15.4±0.5 21.6 ± 1.1
16.7± 1.2 19.2 ± 1.1 21.6 ± 0.8 26.0
4/4 (100) 10/10 (100) 39/40 (98) 9/10 (90)
18/18 (100) 22/24 (92) 31/44 (71) 1/14 (7)
NS NS .001 .001
20.7 ± 0.3
19.0 ± 1.2
6/6 (l00)
5/6 (83)
NS
Macrophages 4 6 8
10
Fibroblasts 10
NOTE. NRS. IRS, normal or immune rabbit serum; NS, not significant. * Duration of in vitro incubation of T. pallidum with rabbit peritoneal macrophagesor testicular fibroblasts. t Fisher's exact test; applies to differencesin proportion of total sites developing lesions.
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o 2 4 6 8 10 Incubation with Macrophages (hours)
71
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Discussion
pallidum. Recent freeze-fracture electron microscopic analyses [37, 38] suggest that this organism has few outer membrane proteins (TROMP), and therefore, extended incubation with immune serum may be necessary to allow sufficient numbers of opsonic antibodies to bind to the organism to promote phagocytosis. However, seemingly contradictory evidence has been provided by Blanco et al. [39], who show rapid binding of antibody to intact T. pallidum. It may be necessary, though, for aggregation of surface antigens to occur in order to trigger phagocytosis, as suggested by these authors for complement-mediated killing. The identity of TROMP molecules has not been reported, so nothing is known yet about when antibodies to these proteins develop. We are currently examining the time course in which antibody specificities that promote phagocytosis and killing of T. pal/idum develop in infected rabbits. The prolonged period required for phagocytosis to occur in the presence of immune serum in the present study is consistent with previous reports [13, 14]. Furthermore, our observation that killing is evident at 10 h of incubation but has not yet occurred at 8 h, despite evidence for substantial phagocytosis by immunofluorescence staining at 8 h, suggests that lysosomal fusion and successful killing may require up to 2 h after ingestion. Although our data reveal that a proportion of un opsonized treponemes were phagocytized in vitro, the number oftreponemes ingested and the number ofmacrophages that are actually phagocytic in vivo are probably too limited, in the absence of specific antibody, to successfully eradicate all organisms early in infection. It is also possible that the low numbers of treponemes phagocytized in the absence of specific antibody may not be killed because of the inability to stimulate an effective oxidative burst. Brett and Butler [40] found that live Mycobacterium lepraemurium were poorly phagocytized in the absence ofimmune serum, and they suggested that phagocytosis of fewer than three or four organisms might be insufficient to trigger an oxidative burst. When the bacteria were preincubated in immune serum, both phagocytosis and the oxidative burst were enhanced. A similar failure to trigger an oxidative burst in the absence of specific antibody was found to occur with Salmonel/a tvphimurium [41] and the intracellular organism Toxoplasma gondii [42]. Recently, some of the microbicidal peptides, defensins, were shown to immobilize and kill T. pal/idum in vitro [43]. Immunoperoxidase staining of defensins in rabbit testicular lesions detected their presence at days 10-16 after T. pallidum infection, concurrent with bacterial clearance. Although neutrophils were identified as a source of defensins [44], a role for this or a similar mechanism of treponemicidal activity by macrophages cannot be precluded. Reports of the production of soluble factors by treponemal antigen-stimulated spleen cell populations, including purified adherent cells, which can immobilize, kill, and lyse T. pal/idum [15,
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These experiments provide direct evidence for the ability ofmacrophages to successfully kill T. pallidum; phagocytosis and treponemicidal activity were dependent on the presence of opsonic antibody. Indirect evidence of the macrophage's ability to kill treponemes has been accumulating for years. Light and electron microscopic evaluations of syphilitic lesions have confirmed that large numbers of macrophages infiltrate the initial site of infection subsequent to T cell infiltration [1-3], and intact and deteriorating treponemes have been identified within macrophage vacuoles [9, 11, 12]. Rapid clearing oftreponemes after apparent phagocytosis by macrophages has also been observed on reinfection of chancre-immune animals [24]. Early studies of rabbits injected with agents that stimulate phagocytosis, such as lecithin or trypan blue, showed more rapid resolution of active syphilitic lesions than in untreated animals [25]. Wicher et al. [26] demonstrated a significant increase in nitroblue tetrazolium reduction by peripheral blood leukocytes from animals infected with T. pal/idum for 5-40 days compared with normals, suggesting increased phagocytic activity. Attempts to alter the course of experimental syphilis by activating macrophages with Mycobacterium bovis [27-30] or Propionibacterium aenes [31] have been generally unsuccessful, although occasional acceleration of lesion development has been described [29, 30]. Azadegan et al. [32] did show that hamsters vaccinated with bacille Cal metre-Guerin were more resistant to challenge with Treponema pallidum subspecies endemieum. Infection with T. pal/idum also activates macrophages in vivo sufficiently to provide increased resistance to Listeria monocytogenes infection [33, 34]. We [35] demonstrated earlier that lymphocytes from T. pallidum-infected rabbits produce macrophage-activating factor (MAF) in response to stimulation by treponemal antigens, whereas normal rabbit lymphocytes fail to produce MAF. Thus, numerous studies have demonstrated that macrophage activity is an important factor in determining the clinical course of syphilitic infection, but this is the first report to confirm that macrophages do, in fact, possess the ability to kill treponemes. Although our demonstration does not unequivocally mean that these mechanisms are operable in vivo, the time frame in which antibody develops, macrophages infiltrate the local site, and organisms are cleared [1, I 1] strongly suggests that they are. Seeding to peripheral sites causing persistent infection probably occurs early in disease, before the development of opsonic antibody. In vitro studies have measured phagocytosis of T. pal/idum by immunofluorescence staining [13, 14] and 51Cr labeling [36]. Both techniques reveal the importance of opsonic antibody for facilitating ingestion, and in most cases phagocytosis proceeds slowly compared with that ofother bacteria. The lengthy incubation time and the requirement for antibody may be -due to the relatively inert nature of the surface of T.
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very rapid clearance of organisms due to the massive cellular infiltration, explaining the relative paucity of organisms in clinically recognized gummas.
Acknowledgments
We thank Jeanne Shaffer and Christa Castro for technical assistance and Sally Post for secretarial help.
References I. Lukehart SA, Baker-Zander SA, Lloyd RMC, Sell S. Characterization of lymphocyte responsiveness in early experimental syphilis. II. Nature of cellular infiltration and Treponema pallidum distribution in testicular lesions. J Immunol 1980; 124:461-7. 2. Sell S, Baker-Zander SA, Lloyd RMe. T cell hyperplasia oflymphoid tissues of rabbits infected with Treponema pallidum: evidence for a vigorous immune response. Sex Transm Dis 1980;7:74-84. 3. Bjerke JR, Krogh HK, Matre R. In situ identification of mononuclear cells in cutaneous infiltrates in discoid lupus erythematosus, sarcoidosis and secondary syphilis. Acta Derm Venereol (Stockh) 1981;61 :371-80. 4. Pearce L, Rosahn PD. The cellular reaction in experimental syphilis. Supravital and fixed material. Proc Soc Exp Bioi Med 1931;28: 654-6. 5. Mercer ST. Preliminary observations on human blood in early syphilis by the supravital method. Proc Soc Exp BioI Med 1931 ;28: 1033-5. 6. Rosahn PO, Pearce L. The blood cytology in untreated and treated syphilis. Am J Med Sci 1934;187:88-100. 7. Lowenstein L. The leucocytes in early acute experimental syphilis in rabbits. Am J Syphilis NeuroI1935;19:39-47. 8. Turner TB, Wright DJM. Lymphadenopathy in early syphilis. J Pathol 1973;110:305-8. 9. Sell S, Baker-Zander SA, Powell He. Experimental syphilitic orchitis in rabbits: ultrastructural appearance of Treponema pal/idum during phagocytosis and dissolution by macrophages in vivo. Lab Invest 1982;46:355-64. 10. Jepson OB, Hougen KH, Birch-Anderson A. Electron microscopy of Treponema pal/idum Nichols. Acta Pathol Microbiol Scand 1969;74:241-58. 11. Lukehart SA, Baker-Zander SA, Lloyd RM, Sell S. Effect of cortisone administration on host-parasite relationships in early experimental syphilis. J ImmunoI1981;127:1361-8. 12. Baker-Zander SA, Sell S. A histopathologic and immunologic study of the course of syphilis in the experimentally infected rabbit. Am J PathoI1980;101:387-414. 13. Lukehart SA, Miller IN. Demonstration of the in vitro phagocytosis of Treponema pallidum by rabbit peritoneal macrophages. J Immunol 1978; 121:20 14-24. 14. Alder JD, Daugherty N, Harris ON, Liu H, Steiner BM, Schell RF. Phagocytosis of Treponema pallidum pertenue by hamster macrophages on membrane filters. J Infect Dis 1989; 160:289-97. 15. Podwinska J. Identification of cells producing antitreponemal lymphotoxin (ATL). Arch Immunol Ther Exp (Warsz) 1987;35:63-70. 16. Fitzgerald TJ, Elmquist BJ. Soluble factors from rabbit spleen cells kill and lyse Treponema pal/idum in vitro. Can J Microbiol 1990;36:711-7. 17. Larsen SA, Hunter EF, Kraus SJ, eds. A manual of tests for syphilis. Washington, DC: American Public Health Association, 1990. 18. Lukehart SA, Baker-Zander SA, Sell S. Characterization oflymphocyte responsiveness in early experimental syphilis. I. In vitro response to
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16] suggest some mechanism of extracellular killing may occur. However, it is highly unlikely that macrophages used in our study secreted extracellular treponemicidal factors, because these factors required prolonged incubation (24-96 h) and ~ I 0 times more cells for production and were not made by normal cells [IS, 16]. The apparent phagocytosis observed in the absence of exogenous IRS may be explained by the observation that proteose peptone-elicited macrophages exhibit a slight degree of nonspecific activation over and above that seen in resting macrophages and that this level might be sufficient to permit the limited phagocytosis of unopsonized treponemes. Alder et al. [14] reported that proteose peptone-induced hamster peritoneal macrophages have an increased ability to phagocytize heat-killed Treponema pallidum subspecies pertenue in the absence ofimmune serum compared with resident macrophages. Alternatively, endogenous rabbit immunoglobulin or C3b known to coat freshly extracted organisms [45] may opsonize T. pallidum adequately to promote limited phagocytosis, particularly over the prolonged (lO h) incubation period in our studies. In conclusion, the evidence provided in this study supports an active role for macrophage-mediated events in early syphilitic infection. One plausible scenario for the development of the immune response to infection with T. pallidum may be the following: After initial penetration of the invading organisms at the primary site, some phagocytosis by wandering macrophages occurs in the absence of immune serum. These macrophages and other antigen-presenting cells (e.g., dendritic cells) process and present treponemal antigens to helper T cells, which in turn secrete lymphokines (e.g., interleukin-2) to up-regulate the T cell response. Subsets of helper T cells provide help for B cells in antibody production and produce macrophage activating factors (e.g., interferon-v). The subsequent development of specific antibody allows opsonization of T. pallidum, facilitating widespread ingestion and destruction of T. pallidum by recruited and activated macrophages, leading to the rapid clearance of most bacteria at the primary site of infection. However, early dissemination of low numbers oftreponemes to peripheral sites permits persistence ofinfection. The successful evasion of the host's defenses by a few organisms, despite the ability oflocal macrophages to phagocytize and kill the vast majority of treponemes, may be due to the relatively inert nature of the treponemal surface. It may be that a "critical mass" oftreponemes is required to trigger the successful T cell and macrophage infiltration seen in primary syphilis. Propagation of disseminated organisms to that "critical mass" may result in the cellular infiltration and consequent lesion development of the secondary stage of syphilis. Once cellular infiltration is triggered, bacterial clearance occurs and the rash resolves without therapy. A similar trigger mechanism may occur in benign tertiary syphilis, with a
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45.
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to infection with Treponema pallidum ssp. endemicum in hamsters. Reg Immunol 1988;1:3-8. Schell RF, Musher DM. Detection of nonspecific resistance to Listeria monocytogenes in rabbits infected with Treponema pa/lidum. Infect Immun 1974;9:658-62. Schell R, Musher D, Jacobson K, Schwethelm P. Induction ofacquired cellular resistance following transfer of thymus-dependent lymphocytes from syphilitic rabbits. J ImmunoI1975;114:550-3. Lukehart SA. Activation of macrophages by products of lymphocytes from normal and syphilitic rabbits. Infect Immun 1982;37:64-9. Metzger M, Michalska E. Treponema pal/idum opsonophagocytic test. Arch Immunol Ther Exp 1974;22:745-58. Radolf JD, Norgard MV, Schulz WW. Outer membrane ultrastructure explains the limited antigenicity of virulent Treponema pallidum. Proc Natl Acad Sci USA 1989;86:2051-5. Walker EM, Zampighi GA, Blanco DR, Miller IN, Lovett MA. Demonstration of rare protein in the outer membrane of Treponema pa/lidum subsp. pa/lidum by freeze-fracture analysis. J Bacteriol 1989; 171:5005-11. Blanco DR, Champion CI, Miller IN, Lovett MA. Complement activation limits the rate of in vitro treponemicidal activity and correlates with antibody-mediated aggregation of Treponema pallidum rare outer membrane protein. J ImmunoI1990;144:1914-21. Brett SJ, Butler R. Interactions of Mycobacterium lepraemurium with resident peritoneal macrophages; phagocytosis and stimulation of the oxidative burst. Clin Exp Immunol 1988;71 :32-8. Yamada Y, Saito H, Tomiaka H, Jidai J. Relationship between the susceptibility of various bacteria to active oxygen species and to intracellular killing by macrophages. J Gen Microbiol 1987; 133: 2015-21. Wilson CB, Tsai V, Remington JS. Failure to trigger the oxidative metabolic burst by normal macrophages. Possible mechanism for survival of intracellular pathogens. J Exp Med 1980; 151:328-46. Borenstein LA, Selsted ME, Lehrer RI, Miller IN. Antimicrobial activity of rabbit leukocyte defensins against Treponema pallidum subsp. pa/lidum. Infect Immun 1991;59: 1359-67. Borenstein LA, Ganz T, Sell S, Lehrer RI, Miller IN. Contribution of rabbit leukocyte defensins to the host response in experimental syphilis. Infect Immun 1991;59: 1368-77. Alderete JF, Baseman JB. Surface-associated host proteins on virulent Treponema pa/lidum. Infect Immun 1979;26:1048-56.
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25.
Baker-Zander and Lukehart
Macrophage-Mediated Killing of Opsonized Treponema pallidum Sharon A. Baker-Zander and Sheila A. Lukehart
Department of Medicine, University of Washington School ofMedicine, Seattle
Macrophages are thought to be responsible for the rapid clearance of Treponema pal/idum subspecies pal/idum from early syphilitic lesions. While studies in vivo and in vitro strongly support this hypothesis, the ability of macrophages to kill T. pal/idum has not been demonstrated. In natural and experimental syphilis infection, lymphocyte and macrophage infiltration in lesions [1-3] and increased numbers of monocytes in peripheral blood [4-7] and macrophages in other lymphoid organs [8] have been well characterized. Light and electron microscopic evaluation oflesion biopsies detect both intact and degenerating treponemes in vacuoles within macrophages, suggesting active phagocytosis and dissolution of T. pal/idum [1, 2, 9, 10]. We have previously demonstrated T. pallidum antigens within macrophages in healing primary lesions in the rabbit model [9,11,12]. Lukehart and Miller [13] documented active phagocytosis of viable T. pallidum by proteose peptone-induced normal rabbit peritoneal macrophages in vitro; organisms were identified within vacuoles by immunofluorescence staining and electron microscopy. Phagocytosis was significantly enhanced by opsonization with immune serum, although a role for complement was not demonstrated. More recently, Alder et a1. [14] confirmed treponemal phagocytosis using hamster peritoneal macrophages and a different subspecies of T. pallidum attached to polycarbonate filters. Again, phagocytosis was increased with immune serum and proceeded slowly. In neither study, however, was the actual killing of organisms by macrophages examined. Some evidence for cell-mediated extracellular treponemiReceived I July 1991; revised 23 September 1991. Grant support: National Institutes of Health (AI-18988). Reprints or correspondence: Sharon A. Baker-Zander, Department of Medicine/Infectious Diseases, ZA-20, Harborview Medical Center, 325 Ninth Ave., Seattle, WA 98104. The Journal ofInfectious Diseases 1992;165:69-74 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6501-0008$01.00
cidal mechanisms also exists. Spleen cell preparations cultured in the presence oftreponemal antigen produce soluble factors that reportedly immobilize and kill T. pallidum [15, 16]. The present report documents killing of T. pal/idum after ingestion by macrophages in vitro.
Materials and Methods Animals. Adult male New Zealand white rabbits were obtained from R & R Rabbitry (Stanwood, WA) and were housed individually at 18-20°C and given antibiotic-free food and water. Rabbits were examined on receipt for serologic evidence of infection with Treponema paraluiscuniculi by the VDRL and fluorescent treponemal antibody-absorbed (FTA-ABS) tests. These tests were done as recommended by the Centers for Disease Control [17] with modifications for use with rabbit sera [11]. Organisms. T. pallidum pal/idum (Nichols strain) was maintained by intratesticular passage in rabbits as previously described [18]. For experiments, organisms were extracted from infected testes in sterile medium 199 (M 199). Gross testicular debris was sedimented by low-speed centrifugation (350 g for 7 min), and organisms in the supernatant were enumerated by darkfield microscopy and diluted in M 199 to 2 X 107/rnl for immunofluorescence studies or 2 X 105 /ml for the in vitro killing assay. Sera. Pools of normal rabbit sera (NRS, VDRL and FTAABS nonreactive) and immune rabbit sera (IRS, from rabbits infected with T. pal/idum either intratesticularly or intradermally for 2-10 months) were prepared. Blood was collected from the medial ear artery into sterile glass tubes and allowed to clot at 4°C overnight. Tubes were spun at 750 g, and the serum was collected, pooled, and filter-sterilized. Pools were then heatinactivated at 56°C for 30 min, dispensed in 3-ml aliquots, and stored frozen at -20°C until use. Specific antitreponemal antibodies in the IRS pool were evaluated by titration in the FTAABS test and by immunoblot against treponemal antigens as described elsewhere [19]. Pooled human syphilitic serum (IHS) was prepared from
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The ability of proteose peptone-induced normal rabbit peritoneal macrophages to kill Treponema pallidum subspecies pallidum in vitro is demonstrated. Treponemes and 10% heated immune or normal sera were incubated with macrophages at a ratio of 1:200. After 2-10 h of incubation, these mixtures were injected intradermally at duplicate sites on normal rabbits. Maximal killing (failure to develop lesions) was seen at 10 h of incubation with immune serum: Only 7% (1/14) of lesions developed compared with 90% (9/10) after incubation in the presence of normal serum (P < .001). Maximal phagocytosis (detected by immunofluorescence) occurred by 8 h in the presence of immune serum, when 90% of macrophages had ingested treponemes. At this point, however, 70% of lesion sites from macrophages incubated with treponemes and immune serum still developed, suggesting that effective killing may require at least 2 h after phagocytosis.
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were counted for each coverslip. The reader was blinded to the identity of each specimen. Data are expressed as mean percentage of cells with labeled vacuoles. Control cultures omitting T. pal/idum were routinely examined in parallel and averaged 1%2% positive. Killing assay. Killing after phagocytosis was evaluated by determining the absence or delay of lesion development after inoculation of T. pal/idum (both cell-associated and extracellular treponemes) after incubation for various times with macrophages or fibroblasts. Macrophage and fibroblast cultures (sixwell plates) were rinsed three times with warm M 199, and 1.9 ml of fresh M 199 supplemented with glutamine and containing either 20% NRS or 10% NRS and 10% IRS (but no antibiotics) was added to the cultures. Freshly extracted T. pal/idum ( 100 JLI ofa suspension of2 X 105 treponemes/rnl) were then added to each culture, yielding a final macrophage-to-treponeme ratio of 200: 1. Cultures were incubated anaerobically as described earlier at 37°C to promote treponemal survival. At various times up to 12 h, macrophages were scraped from the plate surface into the culture medium using a cell scraper, and macrophagetreponeme mixtures were inoculated intradermally at duplicate sites into the clipped backs of seronegative normal rabbits (100JLl vel/site, equal to 103 treponemes initially added in culture). Rabbits were observed for lesion development for 30 days; the absence or delay oflesion development was a direct reflection of decreased numbers of viable T. pal/idum. Five separate macrophage preparations, inoculated into a total of 19 rabbits, were examined. Data analysis. The means ± SE were calculated forimmunofluorescence data; comparisons between groups were done by analysis of variance. Fisher's exact test was used to compare the proportion of lesions that developed in each group. Statistical significance was defined as P < .05.
Results Evaluation of antitreponemal antibodies in NRS and IRS pools used for opsonization. Pooled IRS was evaluated in the FTA-ABS test using serial twofold dilutions of antibody in PBS after initial 1:5 dilution in sorbent. The highest dilution that still gave 2+ fluorescence was I: 1024. The NRS pool was nonreactive in the FTA-ABS test. Both sera were examined by immunoblot against SDSPAGE-separated T. pallidum antigens. The NRS pool diluted 1: 100 failed to detect any treponemal antigens, whereas the IRS pool detected a full complement oftreponemal antigens described previously [19], even at a dilution of 1:500. Phagocytosis of T. pal/idum by peritoneal macrophages. The degree of phagocytosis of viable T. pal/idum by proteose peptone-induced normal peritoneal macrophages in culture was determined by immunofluorescence staining using IHS and FITC-conjugated anti-human IgG. Ingestion in the presence of 20% NRS was compared with that in the presence of 10% NRS plus 10% IRS over a 10-h period (figure 1). Maximal phagocytosis was reached by 8 h of incubation in IRS,
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serum specimens collected from patients diagnosed with primary, secondary, or latent syphilis. Sera were heat-inactivated, dispensed, and stored as above. Rabbit fibroblasts. To provide a control for treponemal survival in the presence ofa nonphagocytic cell line, primary rabbit testicular fibroblast cultures were derived from normal testicular tissue by the method of Fitzgerald et al. [20]. Testes were minced and digested in 0.25% trypsin and 0.0 I% EDTA in PBS (pH 7.8) for 15 min on a magnetic stirrer. The resulting mixture was centrifuged at 400 g for 10 min, and the pellet was washed once in Eagle's MEM with Hanks' salts (MEM-HS) to remove the trypsin. The pellet was suspended in MEM-HS containing 20 mM HEPES, 10% fetal bovine serum, 1% 50X amino acids, 2% 100X vitamins, I%glutamine, and I%penicillin and streptomycin and dispensed into T-150 flasks (Costar, Cambridge, MA). Cultures were maintained at 37°C in 5% CO 2 and air. Medium was changed three times weekly, and fibroblasts were subcultured when confluent. One day before macrophage killing assays, confluent fibroblast cultures were trypsinized, washed twice in complete MEMHS, counted by hemocytometer, and adjusted to 105-106 cells/ ml. Fibroblasts were dispensed in six-well cluster plates (2 ml/well: Costar) and cultured overnight. Preparation ofmacrophages. Rabbit peritoneal macrophages were induced by intraperitoneal injection of 10 ml of 15%sterile proteose peptone 4 days before harvesting by peritoneal lavage with balanced salt solution (BSS) containing heparin (10 units/ ml). Cells were washed three times in cold BSS, counted by hemocytometer, and adjusted to 2 X 106/rnl in M 199 supplemented with 20% heat-inactivated NRS, penicillin, streptomycin, and glutamine. Macrophages were dispensed in 6- or 24well cluster tissue culture plates (2 or 0.5 rnl/well; Costar) and incubated for 2 h at 37°C in 5% CO 2 • Nonadherent cells were removed by mild agitation, fresh medium was added, and the cultures were incubated overnight. Before incubation with treponemes, cultures were washed to remove residual antibiotics. Phagocytosis. For immunofluorescent detection oftreponemal antigens after phagocytosis, macrophages were cultured in 24-well plates (0.5 mljwell) containing sterile glass coverslips in an anaerobic environment generated by anaerobic system envelopes (Difco, Detroit) in Gaspak jars (BBL Microbiology Systems, Cockeysville, MD). At various times after coculture with 107 freshly extracted treponemes, coverslips were rinsed with warm M 199 and fixed in 95% ethanol. Fixed cells on coverslips were rehydrated with PBS for 10 min at room temperature, then incubated with IHS (diluted 1:50 in PBS containing 1% bovine serum albumin) for 30 min at 37°C. After this incubation, coverslips were washed three times with PBS and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-human IgG (Zymed Laboratories, San Francisco) diluted I: 1000 in PBS containing 2% Tween 80 for 30 min at 37°C. Coverslips were again washed three times in PBS, allowed to air dry, and inverted on microscope slides with fluorescence mounting medium. Coverslips were examined under a Zeiss fluorescence microscope for the presence of fluorescein-labeled treponemal antigen in vacuoles within macrophages. Triplicate specimens were prepared for each time point or condition, and 100 macrophages
1ID 1992; 165 (January)
Macrophage Killing of T. pallidum
110 1992; 165 (January)
100-r---------------------. IRS 0)
c
~
80
CD
0)
c
-;;e:
CD:::J
O)~ «S:::::: .s::tU Q.Q"
eJ..,: o
60 NRS
40
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0
20
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0
Figure 1.
Rate of phagocytosis of Treponema pal/idum by normal rabbit peritoneal macrophages in the presence of normal (NRS) or immune rabbit serum (IRS). Triplicate cultures ofmacrophages were incubated with viable T. pal/idum for various times and fixed and stained by immunofluorescence for treponemal antigens. Data points represent mean ± SE percentage of macrophages containing fluorescent-stained treponemes. Differences between NRS and IRS were significant (P < .0 I) by 2 h.
when >90% of macrophages contained ingested fluorescent treponemal antigens. At this time, fewer than 45% of macrophages incubated with treponemes and NRS contained any treponemal antigens (P < .0 I, compared with IRS). Killing after in vitro phagocytosis of T. pallidum. The ability of rabbit peritoneal macrophages to successfully kill ingested T. pallidum is shown in table I. Maximal killing was seen after 10 h ofincubation; only I (7%) of 14 lesions developed from macrophages incubated with treponemes and 10% IRS, compared with 9 (90%) of 10 after incubation with NRS (P < .001). The single lesion that developed in the IRS group was delayed 4.4 days compared with the corresponding NRS
Table 1.
control (26 days for IRS vs. 21.6 ± 1.1 days for NRS). This indicates that only a small portion ofthe original treponemes in this one sample survived the 10-h incubation. The mean time to lesion development after 10 h of incubation with macrophages and NRS was comparable to that of treponemes and fibroblasts incubated in either NRS or IRS (20.7 and 19.0 days, respectively), suggesting that no macrophagemediated killing of T. pal/idum occurred in the presence ofNRS. In this bacterial killing assay, extracellular replication of T. pallidum is not a confounding issue, even though the incubation times were as long as 10 h, because the organism's maximum generation time is 33 h in vivo [21] and is estimated to be often slower in vitro [22, 23]. Virtually no killing was detected after only 4 h of incubation, but an increasing delay in lesion development and fewer numbers of lesions observed thereafter (compared with NRS) suggests that progressively more killing occurred as incubation times were increased. Effects ofculture conditions and incubation time on T. pallidum survival. To demonstrate that the loss ofinfectivity of treponemal suspensions was not due to in vitro incubation conditions alone, cultures ofrabbit testicular fibroblasts were incubated with T. pallidum under identical experimental conditions. Treponemal survival was measured as mean time to onset oflesion development after intradermal inoculation of rabbits (table 1). After a 10-h incubation in the presence of decomplemented NRS, lesions were observed at all sites (6/6) in a mean time of20.7 ± 0.3 days; injection ofcultures incubated in IRS yielded 5 (83.3%) of 6 lesions in a mean time of 19.0 ± 1.2 days. This delay of -- 6 days (beyond the expected onset of lesion development of 13 days if all 103 treponemes had survived) probably reflects a loss oftreponemal viability due to incubation conditions equivalent to 1 log [21]. No treponemicidal effects of incubation in IRS compared with NRS were observed with fibroblasts.
Demonstration of intracellular killing of T. pallidum. Days to lesion development (mean ± SE)
Hours*
Lesions/total sites (%)
NRS
IRS
NRS
IRS
pt
15.5 ± 1.9 15.7±0.9 15.4±0.5 21.6 ± 1.1
16.7± 1.2 19.2 ± 1.1 21.6 ± 0.8 26.0
4/4 (100) 10/10 (100) 39/40 (98) 9/10 (90)
18/18 (100) 22/24 (92) 31/44 (71) 1/14 (7)
NS NS .001 .001
20.7 ± 0.3
19.0 ± 1.2
6/6 (l00)
5/6 (83)
NS
Macrophages 4 6 8
10
Fibroblasts 10
NOTE. NRS. IRS, normal or immune rabbit serum; NS, not significant. * Duration of in vitro incubation of T. pallidum with rabbit peritoneal macrophagesor testicular fibroblasts. t Fisher's exact test; applies to differencesin proportion of total sites developing lesions.
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o 2 4 6 8 10 Incubation with Macrophages (hours)
71
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Baker-Zander and Lukehart
Discussion
pallidum. Recent freeze-fracture electron microscopic analyses [37, 38] suggest that this organism has few outer membrane proteins (TROMP), and therefore, extended incubation with immune serum may be necessary to allow sufficient numbers of opsonic antibodies to bind to the organism to promote phagocytosis. However, seemingly contradictory evidence has been provided by Blanco et al. [39], who show rapid binding of antibody to intact T. pallidum. It may be necessary, though, for aggregation of surface antigens to occur in order to trigger phagocytosis, as suggested by these authors for complement-mediated killing. The identity of TROMP molecules has not been reported, so nothing is known yet about when antibodies to these proteins develop. We are currently examining the time course in which antibody specificities that promote phagocytosis and killing of T. pal/idum develop in infected rabbits. The prolonged period required for phagocytosis to occur in the presence of immune serum in the present study is consistent with previous reports [13, 14]. Furthermore, our observation that killing is evident at 10 h of incubation but has not yet occurred at 8 h, despite evidence for substantial phagocytosis by immunofluorescence staining at 8 h, suggests that lysosomal fusion and successful killing may require up to 2 h after ingestion. Although our data reveal that a proportion of un opsonized treponemes were phagocytized in vitro, the number oftreponemes ingested and the number ofmacrophages that are actually phagocytic in vivo are probably too limited, in the absence of specific antibody, to successfully eradicate all organisms early in infection. It is also possible that the low numbers of treponemes phagocytized in the absence of specific antibody may not be killed because of the inability to stimulate an effective oxidative burst. Brett and Butler [40] found that live Mycobacterium lepraemurium were poorly phagocytized in the absence ofimmune serum, and they suggested that phagocytosis of fewer than three or four organisms might be insufficient to trigger an oxidative burst. When the bacteria were preincubated in immune serum, both phagocytosis and the oxidative burst were enhanced. A similar failure to trigger an oxidative burst in the absence of specific antibody was found to occur with Salmonel/a tvphimurium [41] and the intracellular organism Toxoplasma gondii [42]. Recently, some of the microbicidal peptides, defensins, were shown to immobilize and kill T. pal/idum in vitro [43]. Immunoperoxidase staining of defensins in rabbit testicular lesions detected their presence at days 10-16 after T. pallidum infection, concurrent with bacterial clearance. Although neutrophils were identified as a source of defensins [44], a role for this or a similar mechanism of treponemicidal activity by macrophages cannot be precluded. Reports of the production of soluble factors by treponemal antigen-stimulated spleen cell populations, including purified adherent cells, which can immobilize, kill, and lyse T. pal/idum [15,
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These experiments provide direct evidence for the ability ofmacrophages to successfully kill T. pallidum; phagocytosis and treponemicidal activity were dependent on the presence of opsonic antibody. Indirect evidence of the macrophage's ability to kill treponemes has been accumulating for years. Light and electron microscopic evaluations of syphilitic lesions have confirmed that large numbers of macrophages infiltrate the initial site of infection subsequent to T cell infiltration [1-3], and intact and deteriorating treponemes have been identified within macrophage vacuoles [9, 11, 12]. Rapid clearing oftreponemes after apparent phagocytosis by macrophages has also been observed on reinfection of chancre-immune animals [24]. Early studies of rabbits injected with agents that stimulate phagocytosis, such as lecithin or trypan blue, showed more rapid resolution of active syphilitic lesions than in untreated animals [25]. Wicher et al. [26] demonstrated a significant increase in nitroblue tetrazolium reduction by peripheral blood leukocytes from animals infected with T. pal/idum for 5-40 days compared with normals, suggesting increased phagocytic activity. Attempts to alter the course of experimental syphilis by activating macrophages with Mycobacterium bovis [27-30] or Propionibacterium aenes [31] have been generally unsuccessful, although occasional acceleration of lesion development has been described [29, 30]. Azadegan et al. [32] did show that hamsters vaccinated with bacille Cal metre-Guerin were more resistant to challenge with Treponema pallidum subspecies endemieum. Infection with T. pal/idum also activates macrophages in vivo sufficiently to provide increased resistance to Listeria monocytogenes infection [33, 34]. We [35] demonstrated earlier that lymphocytes from T. pallidum-infected rabbits produce macrophage-activating factor (MAF) in response to stimulation by treponemal antigens, whereas normal rabbit lymphocytes fail to produce MAF. Thus, numerous studies have demonstrated that macrophage activity is an important factor in determining the clinical course of syphilitic infection, but this is the first report to confirm that macrophages do, in fact, possess the ability to kill treponemes. Although our demonstration does not unequivocally mean that these mechanisms are operable in vivo, the time frame in which antibody develops, macrophages infiltrate the local site, and organisms are cleared [1, I 1] strongly suggests that they are. Seeding to peripheral sites causing persistent infection probably occurs early in disease, before the development of opsonic antibody. In vitro studies have measured phagocytosis of T. pal/idum by immunofluorescence staining [13, 14] and 51Cr labeling [36]. Both techniques reveal the importance of opsonic antibody for facilitating ingestion, and in most cases phagocytosis proceeds slowly compared with that ofother bacteria. The lengthy incubation time and the requirement for antibody may be -due to the relatively inert nature of the surface of T.
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Macrophage Killing of T. pallidum
very rapid clearance of organisms due to the massive cellular infiltration, explaining the relative paucity of organisms in clinically recognized gummas.
Acknowledgments
We thank Jeanne Shaffer and Christa Castro for technical assistance and Sally Post for secretarial help.
References I. Lukehart SA, Baker-Zander SA, Lloyd RMC, Sell S. Characterization of lymphocyte responsiveness in early experimental syphilis. II. Nature of cellular infiltration and Treponema pallidum distribution in testicular lesions. J Immunol 1980; 124:461-7. 2. Sell S, Baker-Zander SA, Lloyd RMe. T cell hyperplasia oflymphoid tissues of rabbits infected with Treponema pallidum: evidence for a vigorous immune response. Sex Transm Dis 1980;7:74-84. 3. Bjerke JR, Krogh HK, Matre R. In situ identification of mononuclear cells in cutaneous infiltrates in discoid lupus erythematosus, sarcoidosis and secondary syphilis. Acta Derm Venereol (Stockh) 1981;61 :371-80. 4. Pearce L, Rosahn PD. The cellular reaction in experimental syphilis. Supravital and fixed material. Proc Soc Exp Bioi Med 1931;28: 654-6. 5. Mercer ST. Preliminary observations on human blood in early syphilis by the supravital method. Proc Soc Exp BioI Med 1931 ;28: 1033-5. 6. Rosahn PO, Pearce L. The blood cytology in untreated and treated syphilis. Am J Med Sci 1934;187:88-100. 7. Lowenstein L. The leucocytes in early acute experimental syphilis in rabbits. Am J Syphilis NeuroI1935;19:39-47. 8. Turner TB, Wright DJM. Lymphadenopathy in early syphilis. J Pathol 1973;110:305-8. 9. Sell S, Baker-Zander SA, Powell He. Experimental syphilitic orchitis in rabbits: ultrastructural appearance of Treponema pal/idum during phagocytosis and dissolution by macrophages in vivo. Lab Invest 1982;46:355-64. 10. Jepson OB, Hougen KH, Birch-Anderson A. Electron microscopy of Treponema pal/idum Nichols. Acta Pathol Microbiol Scand 1969;74:241-58. 11. Lukehart SA, Baker-Zander SA, Lloyd RM, Sell S. Effect of cortisone administration on host-parasite relationships in early experimental syphilis. J ImmunoI1981;127:1361-8. 12. Baker-Zander SA, Sell S. A histopathologic and immunologic study of the course of syphilis in the experimentally infected rabbit. Am J PathoI1980;101:387-414. 13. Lukehart SA, Miller IN. Demonstration of the in vitro phagocytosis of Treponema pallidum by rabbit peritoneal macrophages. J Immunol 1978; 121:20 14-24. 14. Alder JD, Daugherty N, Harris ON, Liu H, Steiner BM, Schell RF. Phagocytosis of Treponema pallidum pertenue by hamster macrophages on membrane filters. J Infect Dis 1989; 160:289-97. 15. Podwinska J. Identification of cells producing antitreponemal lymphotoxin (ATL). Arch Immunol Ther Exp (Warsz) 1987;35:63-70. 16. Fitzgerald TJ, Elmquist BJ. Soluble factors from rabbit spleen cells kill and lyse Treponema pal/idum in vitro. Can J Microbiol 1990;36:711-7. 17. Larsen SA, Hunter EF, Kraus SJ, eds. A manual of tests for syphilis. Washington, DC: American Public Health Association, 1990. 18. Lukehart SA, Baker-Zander SA, Sell S. Characterization oflymphocyte responsiveness in early experimental syphilis. I. In vitro response to
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16] suggest some mechanism of extracellular killing may occur. However, it is highly unlikely that macrophages used in our study secreted extracellular treponemicidal factors, because these factors required prolonged incubation (24-96 h) and ~ I 0 times more cells for production and were not made by normal cells [IS, 16]. The apparent phagocytosis observed in the absence of exogenous IRS may be explained by the observation that proteose peptone-elicited macrophages exhibit a slight degree of nonspecific activation over and above that seen in resting macrophages and that this level might be sufficient to permit the limited phagocytosis of unopsonized treponemes. Alder et al. [14] reported that proteose peptone-induced hamster peritoneal macrophages have an increased ability to phagocytize heat-killed Treponema pallidum subspecies pertenue in the absence ofimmune serum compared with resident macrophages. Alternatively, endogenous rabbit immunoglobulin or C3b known to coat freshly extracted organisms [45] may opsonize T. pallidum adequately to promote limited phagocytosis, particularly over the prolonged (lO h) incubation period in our studies. In conclusion, the evidence provided in this study supports an active role for macrophage-mediated events in early syphilitic infection. One plausible scenario for the development of the immune response to infection with T. pallidum may be the following: After initial penetration of the invading organisms at the primary site, some phagocytosis by wandering macrophages occurs in the absence of immune serum. These macrophages and other antigen-presenting cells (e.g., dendritic cells) process and present treponemal antigens to helper T cells, which in turn secrete lymphokines (e.g., interleukin-2) to up-regulate the T cell response. Subsets of helper T cells provide help for B cells in antibody production and produce macrophage activating factors (e.g., interferon-v). The subsequent development of specific antibody allows opsonization of T. pallidum, facilitating widespread ingestion and destruction of T. pallidum by recruited and activated macrophages, leading to the rapid clearance of most bacteria at the primary site of infection. However, early dissemination of low numbers oftreponemes to peripheral sites permits persistence ofinfection. The successful evasion of the host's defenses by a few organisms, despite the ability oflocal macrophages to phagocytize and kill the vast majority of treponemes, may be due to the relatively inert nature of the treponemal surface. It may be that a "critical mass" oftreponemes is required to trigger the successful T cell and macrophage infiltration seen in primary syphilis. Propagation of disseminated organisms to that "critical mass" may result in the cellular infiltration and consequent lesion development of the secondary stage of syphilis. Once cellular infiltration is triggered, bacterial clearance occurs and the rash resolves without therapy. A similar trigger mechanism may occur in benign tertiary syphilis, with a
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