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Infection and Immunity, July 2005, p. 4315-4322, Vol. 73, No. 7
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.7.4315-4322.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Chlamydia pneumoniae Augments the Oxidized Low-Density Lipoprotein-Induced Death of Mouse Macrophages by a Caspase-Independent Pathway

Kambiz Yaraei,¹ Lee Ann Campbell,¹ Xiaodong Zhu,² W. Conrad Liles,² Cho-chou Kuo,¹ and Michael E. Rosenfeld^1*

Departments of Pathobiology,¹ Medicine, University of Washington, Seattle, Washington²

Received 9 December 2004/ Returned for modification 3 February 2005/ Accepted 28 February 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Chlamydia pneumoniae is a common respiratory pathogen that is associated with an increased risk of cardiovascular disease. However, the mechanisms by which C. pneumoniae contributes to cardiovascular disease have not been determined yet. C. pneumoniae infection may accelerate the death of cells within atherosclerotic lesions and contribute to the formation of unstable lesions. To test this hypothesis, the impact of C. pneumoniae infection on the death of lipid-loaded mouse macrophages was investigated. It was observed that RAW 264.7 cells are highly susceptible to the toxic effects of oxidized low-density lipoprotein (LDL) and exhibit markers of cell death within 24 h of treatment with as little as 5 µg/ml oxidized LDL. Subsequent infection with either live C. pneumoniae or heat-killed or UV-inactivated C. pneumoniae at a low multiplicity of infection for 24 to 72 h stimulated both additional binding of annexin V and the uptake of propidium iodide. Thus, C. pneumoniae augments the effects of oxidized LDL on cell death independent of a sustained infection. However, unlike oxidized LDL, C. pneumoniae infection does not activate caspase 3 or induce formation of the mitochondrial transition pore or the fragmentation of DNA, all of which are classical markers of apoptosis. Furthermore, primary bone marrow macrophages isolated from mice deficient in Toll-like receptor 2 (TLR-2) but not TLR-4 are resistant to C. pneumoniae-induced death. These data suggest that C. pneumoniae kills cells by a caspase-independent pathway and that the process is potentially mediated by activation of TLR-2.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Chlamydia pneumoniae is a common respiratory pathogen that induces recurrent airway infections, particularly in the elderly and other susceptible individuals. C. pneumoniae is an obligate intracellular pathogen and has a unique growth cycle within cells. It attaches to a cell and is phagocytosed into the cell as elementary bodies. Once inside the cell, the elementary bodies change to reticulate bodies within the first 24 h and multiply. This is followed within the next 24 to 48 h by differentiation back to elementary bodies, which lyse and kill the host cell and propagate the infection (8). C. pneumoniae infects both epithelial cells and macrophages within the lungs, and the infection can be disseminated to other tissues by infected macrophages (8, 33). Because C. pneumoniae is dependent upon the host cell for sustained growth, it is logical to assume that the organism prevents the host cell from undergoing apoptosis until the entire growth cycle has been completed. However, there have been several recent reports suggesting that chlamydiae can either induce apoptosis or prevent apoptosis depending on the conditions of the infection and type of host cell (1, 9, 12-14, 35, 37-40, 46, 48, 49). The exact mechanisms by which C. pneumoniae induces or inhibits apoptosis have not been determined yet.

Infection with C. pneumoniae increases the risk of cardiovascular disease morbidity and mortality. Antibody titers are elevated in individuals with established cardiovascular disease, and C. pneumoniae DNA and protein have been detected within atherosclerotic lesions (8). Sustained infection occurs in endothelial cells, smooth muscle cells, and macrophages, the major cell types within atherosclerotic lesions (15). Macrophage death in atherosclerotic lesions contributes to the formation of the necrotic core and may also contribute to the processes that lead to plaque rupture and destabilization (4, 6, 18, 24, 29). It is still not known precisely what causes the death of macrophages within atherosclerotic lesions and whether protection from cell death prevents plaque destabilization. Most macrophages within atherosclerotic plaques are converted into foam cells by the unregulated accumulation of lipid. Components of oxidized low-density lipoprotein (ox-LDL) and/or unesterified cholesterol kill cultured macrophages by both apoptotic and nonapoptotic pathways (2, 5, 11, 27, 28, 30, 36, 47, 50, 51, 53-55). To date, it is not known whether C. pneumoniae infection kills macrophage-derived foam cells and, in turn, contributes to the destabilization of advanced atherosclerotic lesions.

It has previously been reported that lipid loading of macrophages inhibits infection by C. pneumoniae (7). Thus, foam cells may be resistant to death due to the sustained growth of C. pneumoniae. However, foam cells continue to become activated by C. pneumoniae independent of C. pneumoniae growth and secrete cytokines, such as tumor necrosis factor alpha, that are known to induce apoptosis in many cell types (7). Therefore, whether treatment of oxidized LDL-loaded macrophages with C. pneumoniae has any effect on cell death and, if so, whether this death occurs by an apoptotic, caspase-dependent pathway were investigated. Surprisingly, both live and inactivated C. pneumoniae had an additive effect on death due to oxidized LDL, but the death was not due to a classical apoptotic pathway. Furthermore, primary bone marrow macrophages isolated from mice deficient in Toll-like receptor 2 (TLR-2) but not TLR-4 were resistant to C. pneumoniae-induced death.

Top Abstract Introduction Materials and Methods Results Discussion References

 
RAW 264.7 cells and C. pneumoniae inoculum. RAW 264.7 mouse macrophages (American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum. C. pneumoniae strain AR-39 was propagated in HL cells and purified. The purified chlamydial elementary bodies were suspended in sucrose-phosphate-glutamic acid buffer (0.2 M sucrose, 3.8 mM KH₂PO₄, 6.7 mM Na₂HPO₄, 5 mM L-glutamic acid; pH 7.4) and stored in stock suspensions at –70°C until they were used. The number of chlamydial inclusion-forming units was determined by infecting RAW cells with serially diluted elementary bodies. Chlamydial inclusions were stained with a chlamydial genus-specific monoclonal antibody conjugated with fluorescein isothiocyanate (generated and conjugated in our laboratories) at 72 h postinfection and were counted with a fluorescence microscope. C. pneumoniae was heat inactivated by incubation for 30 min at 56°C in a water bath and was inactivated with UV light by exposure for 30 min to a 30-W UV germicidal light source at a distance of 15 cm (22, 23).

Preparation of bone marrow cells. Twenty-week-old C57BL/6J mice were obtained from the Jackson Laboratory (Bar Harbor, ME). TLR-2^–/– and TLR-4^–/– mice with a C57BL/6J background were kindly provided by Thomas Hawn of the Institute for Systems Biology, Seattle, WA. Four mice were kept in each filter top cage in a modified specific-pathogen-free facility. Mice were fed a regular chow diet. Mice were sacrificed by exsanguination, and the femora were aseptically removed and dissected free of adhering tissues. The bone marrow cells were flushed out by injection of RPMI 1640 medium (Invitrogen, Grand Island, NY) at one end of the bone using a sterile needle. The bone marrow cells that were collected were incubated in a bacteriologic plate in medium containing 50% RPMI 1640 medium, 20% fetal bovine serum, 1% L-glutamine, 1% HEPES, 0.5% penicillin-streptomycin, and 50% L929 cell-conditioned medium. After 7 to 10 days of incubation, cells were harvested and used for the experiments. The experiments were approved by the University of Washington Institutional Animals Care and Use Committee.

Preparation of LDL and induction of foam cells. LDL was isolated from healthy, normolipidemic donors by ultracentrifugation as described previously (41) and was dialyzed against 0.15 M NaCl and 0.05% EDTA, followed by storage under nitrogen. The LDL protein and cholesterol contents were determined by a protein assay (Bio-Rad) and with a cholesterol test kit (Sigma, St. Louis, MO), respectively. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of LDL showed no increase in relative electrophoretic mobility in 2 weeks, indicating that the isolated LDL did not become spontaneously oxidized. The isolated LDL was stored at 4°C and used within 1 week of isolation. LDL concentrations are reported below in micrograms of protein per milliliter. ox-LDL was generated by incubation of LDL (300 µg/ml) in the presence of 5 µmol/liter copper sulfate for 18 h at 37°C. RAW cells and bone marrow macrophages were plated at a concentration of 2.5 x 10⁵ cells/ml in bacteriologic plates. After 24 h, the cells were treated with either 5 µg/ml or 30 µg/ml ox-LDL or 5 µg/ml native LDL (n-LDL) for 24 h. Control cells were not treated.

Inoculation of foam cells with C. pneumoniae. Both lipid-loaded and untreated RAW cells were inoculated with live, heat-killed, and UV-inactivated C. pneumoniae at multiplicities of infection (MOIs) of 0.01, 0.10, and 1.0 and were adsorbed at 35°C in a 5% CO₂ atmosphere for 2 h on a rocker platform. The cells were then cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum without antibiotics for up to 72 h prior to evaluation of markers of cell death. Mouse bone marrow-derived macrophages were inoculated with bacteria at an MOI of 0.01 and treated as described above. Staurosporine (1 µM; Sigma, St.Louis, MO.) was used as a positive control for induction of cell death.

Annexin V staining and propidium iodide uptake. After treatment, the RAW cells and bone marrow macrophages were washed with phosphate-buffered saline, trypsinized (using 1 ml of 2% trypsin in a 5% CO₂ atmosphere at 37°C), and harvested by detaching the cells with fluid by repeated pipetting. The cells were collected by centrifugation and stained with fluorescein isothiocyanate-conjugated annexin V in the presence of Ca²⁺ buffer to measure the amount bound to phosphatidylserine on the outer leaflet of the plasma membrane. Additional cells were treated with propidium iodide (PI) for 10 min in the dark at 25°C for evaluation of membrane integrity according to the manufacturer's instructions (BD Biosciences, Palo Alto, CA). The binding of annexin V and the uptake of PI were evaluated by flow cytometry (FACScan; Becton Dickinson, San Jose, CA) after gaiting of the cells by means of their forward and side scatter properties.

Measurement of activated caspase 3, TUNEL staining, and mitochondrial transition pore formation. To determine whether ox-LDL or C. pneumoniae activated caspase 3, RAW cells were treated with the fluorescent caspase 3 substrate PhiPhiLux (OncoImmunin, Inc., Gaithersburg, MD) according to manufacturer's instructions. The cells were viewed by fluorescence microscopy, the number of cells containing activated caspase 3 was determined for three to five fields, and the results were expressed as the percentage of positive cells. Additional cells were plated on chamber slides (Nalge Nunc International Corp., Naperville, IL). Following treatment, the cells were fixed with 4% paraformaldehyde in phosphate-buffered saline, pH 7.4, for 30 to 45 min at 25°C and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate, and the TUNEL (terminal deoxynucleotide transferase-mediated dUTP nick end labeling) reaction was performed according to the recommendations of the manufacturer (in situ cell death detection kit; Roche, Indianapolis, IN). The number of cells with fragmented DNA that were TUNEL positive and had incorporated fluorescein-labeled nucleotide into DNA during repair was determined with a fluorescence microscope. The data were expressed as the percentage of positive cells in each chamber slide. For evaluation of mitochondrial pore formation, the cells were incubated with 200 nM chloromethyl-x-rosamine (MitoTrackerRed CMX Ros; Molecular Probes, Eugene, Oreg.), and the fluorescence intensity was measured by flow cytometry as described above.

Statistics. Differences in the means for the groups were compared by the two-tailed Student t test assuming equal variances. A P value of <0.05 was considered significant. All experiments included triplicate determinations and were performed at least three times.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Oxidized LDL and C. pneumoniae independently induce externalization of phosphatidylserine and the uptake of propidium iodide in a dose- and time-dependent manner. To determine the optimum doses and time course for investigating the combined effects of ox-LDL and C. pneumoniae on markers of cell death in the RAW cells, the baseline effects of ox-LDL and C. pneumoniae alone in the model system were established. As shown in Fig. 1A, there was both a dose- and time-dependent increase in the binding of annexin V to the cells in response to treatment with ox-LDL. Based on these data, a low dose of ox-LDL (5 µg/ml) for 24 h rather than the higher dose of ox-LDL that induced binding of annexin V in more than 60% of the cells was chosen for all of the subsequent experiments. The next series of experiments focused on determining whether C. pneumoniae alone had any effect on inducing markers of cell death and, if so, what the optimum MOI for all of the subsequent studies was. As shown in Fig. 1B and C, C. pneumoniae infection induced both binding of annexin V and uptake of PI following 24 h of incubation in a dose-dependent manner. Based on these results, the lowest MOI (0.01) was chosen for all subsequent studies. It was also observed that C. pneumoniae that had been inactivated by heat or UV light induced both the binding of annexin V and the uptake of PI at levels comparable to the levels for live C. pneumoniae following 24 h of treatment (Fig. 1B and C).

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FIG. 1. Time course and dose responses for the effects of oxidized LDL and C. pneumoniae on induction of markers of cell death in RAW mouse macrophages. (A) RAW cells were incubated with either a low dose (7.5 µg/ml) or a high dose (30 µg/ml) of oxidized LDL for either 6 h or 24 h and were compared to untreated control cells (No treatment). The percentage of cells that bound fluorescein-labeled annexin V was determined by flow cytometry. The data are the means ± standard deviations of three experiments. An asterisk indicates that the P value is <0.05 for a comparison with cells treated with 7.5 µg/ml ox-LDL and with no-treatment control cells. A dagger indicates that the P value is <0.05 for a comparison with the no-treatment control cells. (B and C) RAW cells were incubated with either live C. pneumoniae (live C.p.), heat-killed C. pneumoniae (heat killed C.p.), or UV light-inactivated C. pneumoniae (UV inactivated C.p.) at different multiplicities of infection for 24 h and compared to untreated control cells (No treatment). The percentage of cells that bound fluorescein-labeled annexin V (B) or took up propidium iodide (C) was determined by flow cytometry. The data are the means ± standard deviations of three experiments. An asterisk indicates that the P value is <0.05 for a comparison with the no-treatment control cells. A dagger indicates that the P value is <0.05 for a comparison with the cells treated with C. pneumoniae at MOIs of 0.01 and 0.1 (B) and at an MOI of 0.01 (C).

Combination of ox-LDL and C. pneumoniae has an additive effect on induction of markers of cell death. As shown in Fig. 2, treatment of the cells with 5 µg/ml of ox-LDL for 24 h followed by infection with live or inactivated C. pneumoniae for 24 to 72 h resulted in a statistically significant increase in the binding of annexin V and the uptake of PI compared to the effects of treatment with 5 µg/ml ox-LDL alone. Again, the inactivated forms of C. pneumoniae had an effect comparable to the effect of live C. pneumoniae. However, with additional time postinfection, there was greater combined induction of the markers of cell death in the cells treated with the live C. pneumoniae than in the cells treated with the inactivated forms (Fig. 2). This presumably reflected the effects of C. pneumoniae growth in the foam cells in which C. pneumoniae infectivity was not prevented by the uptake of lipid. Whether the combination of pretreatment with 5 µg/ml of native LDL and then infection with C. pneumoniae had an additive effect on induction of markers of cell death was also investigated. As expected, native LDL alone had no effect on cell death. Surprisingly, there was again an increase in binding of annexin V and uptake of PI with the combination of n-LDL and C. pneumoniae, although the levels were considerably below the level induced by the combination of ox-LDL and C. pneumoniae (Fig. 3). This may have reflected the capacity of the macrophages to oxidize some of the added n-LDL during the 24-h pretreatment and the known inductive effects of C. pneumoniae on oxidation of LDL (20).

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FIG. 2. Time course of the combined effects of oxidized LDL and C. pneumoniae on induction of markers of cell death in RAW mouse macrophages. (A and B) Control RAW cells were not treated throughout the experiment (No Treatment). Another group of cells were incubated with oxidized LDL (5 µg/ml) for up to 72 h (Ox-LDL). A third group of cells were not treated for the first 24 h and subsequently were incubated with live C. pneumoniae (MOI, 0.01) for up to 72 h (C.p.). The last group of cells were treated with ox-LDL for 24 h and subsequently incubated with live C. pneumoniae for up to 72 h (Ox-LDL + C.p.). The percentage of total cells that bound fluorescein-labeled annexin V (A) or took up propidium iodide (B) at 24, 48, and 72 h postinfection was determined by flow cytometry. The data for all of the cells treated with either ox-LDL alone, ox-LDL plus C. pneumoniae, or C. pneumoniae alone were statistically different at a P value of <0.05 compared to the data for the no-treatment group at all times. The data for cells treated with ox-LDL plus C. pneumoniae were statistically different from the data for the cells treated with ox-LDL alone at 48 and 72 h postinfection (A) and at all times (B) and were different from the data for cells treated with C. pneumoniae alone at all times (A) and at 48 and 72 h (B). The data are the means ± standard deviations of three experiments. (C and D) Control RAW cells were not treated throughout the experiment (No Treatment). Another group of cells were incubated with oxidized LDL (5 µg/ml) for up to 72 h (Ox-LDL). A third group of cells were not treated for the first 24 h and subsequently were incubated with heat-killed C. pneumoniae (MOI, 0.01) for up to 72 h (H.K.C.p.). The last group of cells were treated with ox-LDL for 24 h and subsequently incubated with heat-killed C. pneumoniae for up to 72 h (Ox-LDL + H.K.C.p.). The percentage of total cells that bound fluorescein-labeled annexin V (C) or took up propidium iodide (D) at 24, 48, and 72 h postinfection was determined by flow cytometry. The data for all of the cells treated with either ox-LDL alone or ox-LDL plus heat-killed C. pneumoniae were statistically different at a P value of < 0.05 from the data for the no-treatment group at all times (C and D). The data for cells treated with ox-LDL plus heat-killed C. pneumoniae were statistically different from the data for the cells treated with ox-LDL alone at 24 and 72 h postinfection (A) and at all times (B) and were different from the data for the cells treated with heat-killed C. pneumoniae alone at all times (A) and at 48 and 72 h (D). The data are the means ± standard deviations of three experiments.

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FIG. 3. Time course of the combined effects of native LDL and C. pneumoniae on induction of markers of cell death in RAW mouse macrophages. Control RAW cells were not treated throughout the experiment (Cell only). Another group of cells were incubated with native LDL (5 µg/ml) for up to 72 h (n-LDL). A third group of cells were not treated for the first 24 h and subsequently were incubated with live C. pneumoniae (MOI, 0.01) for up to 72 h (C.p.). The last group of cells were treated with native LDL for 24 h and subsequently incubated with live C. pneumoniae for up to 72 h (n-LDL + C.p.). The percentage of total cells that bound fluorescein-labeled annexin V (upper panel) or took up propidium iodide (lower panel) at 24, 48, and 72 h postinfection was determined by flow cytometry. (Upper panel) An asterisk indicates that the P value is <0.05 for a comparison with the no-treatment control cells. A dagger indicates that the P value is <0.05 for a comparison with the no-treatment control cells and cells treated with n-LDL or C. pneumoniae. A double dagger indicates that the P value is <0.05 for a comparison with no-treatment control cells and cells treated with n-LDL. An arrow with two heads indicates that the P value is <0.05 for a comparison with no-treatment control cells and cells treated with n-LDL plus C. pneumoniae. (Lower panel) An asterisk indicates that the P value is <0.05 for a comparison with the no-treatment control cells, cells treated with C. pneumoniae, and cells treated with n-LDL. A dagger indicates that the P value is <0.05 for a comparison with the no-treatment control cells and cells treated with n-LDL plus C. pneumoniae. A double dagger indicates that the P value is <0.05 for a comparison with no-treatment control cells and cells treated with n-LDL. An arrow with two heads indicates that the P value is <0.05 for a comparison with no-treatment control cells. The data are the means ± standard deviations of results from three experiments.

C. pneumoniae infection does not contribute to caspase-dependent apoptosis in ox-LDL-treated RAW cells. To determine whether ox-LDL and C. pneumoniae induce death by similar mechanisms, the effects on the activation of caspase 3, the formation of the mitochondrial transition pore, and the fragmentation of DNA, all of which are markers of a classical apoptotic process, were investigated. As shown in Fig. 4, treatment with 5 µg/ml of ox-LDL for 24 h activated caspase 3, while infection with C. pneumoniae did not. Consistent with the data described above, treatment with 5 µg/ml ox-LDL stimulated formation of the mitochondrial transition pore. In contrast, infection with C. pneumoniae at an MOI of 0.01 for 24 h did not induce mitochondrial pore formation (Fig. 5). Infection at an MOI of 0.01 also did not induce pore formation up to 72 h postinfection (data not shown). In contrast, at an MOI of 1.0, there was limited induction of the mitochondrial pore at 72 h in about 20% of the infected cells (data not shown). Treatment of the cells with 5 µg/ml of oxidized LDL for 24 to 72 h induced a significant increase in DNA fragmentation (i.e., the percentage of TUNEL-positive cells). In contrast, infection with C. pneumoniae alone did not induce DNA fragmentation, and there was no additive effect of C. pneumoniae in combination with ox-LDL (Table 1).

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FIG. 4. Caspase 3 is activated by oxidized LDL treatment but not by C. pneumoniae infection in RAW cells. RAW cells were treated with oxidized LDL (5 µg/ml) for 24 h (A), were not treated for 24 h and then infected with live C. pneumoniae at an MOI of 0.01 for 72 h (B), or were left untreated throughout the experiment (C). The cells were incubated with the cell-permeable caspase 3 substrate PhiPhiLux, which fluoresces upon activation (OncoImmun, Inc., Gaithersburg, MD). The micrographs are fluorescent micrographs of representative fields of cells (magnification, x100).

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FIG. 5. Oxidized LDL induces formation of the mitochondrial transition pore in RAW cells, while infection with C. pneumoniae does not induce pore formation. RAW cells were either not treated (Control) or treated with a low dose (5 µg/ml) or a high dose (30 µg/ml) of oxidized LDL for 24 h. Additional cells were infected with C. pneumoniae (C.p.) at different MOIs for 24 h. The cells were then incubated with 200 nM chloromethyl-x-rosamine (MitoTrackerRed CMX Ros; Molecular Probes, Eugene, Oreg.), and the fluorescence intensity was measured by flow cytometry. The data are the means ± standard deviations of three experiments. An asterisk indicates that the P value is <0.05 for a comparison with control cells and cells infected with C. pneumoniae at an MOI of 0.01. A dagger indicates that the P value is <0.05 for a comparison with control cells and cells infected with C. pneumoniae at MOIs of 0.01, 0.1, and 1.0. A double dagger indicates that the P value is <0.05 for a comparison with control cells and cells infected with C. pneumoniae at MOIs of 0.01 and 0.1.

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TABLE 1. Effects of oxidized LDL and C. pneumoniae on DNA fragmentation in RAW cells

Bone marrow macrophages from TLR-2-deficient mice are resistant to death due to C. pneumoniae. Both heat-killed and UV-inactivated C. pneumoniae induced markers of cell death in the RAW cells (Fig. 1 and 2), and C. pneumoniae lipopolysaccharide (LPS) and heat shock proteins are known to bind to Toll-like receptors (41). Thus, the capacity of bone marrow macrophages isolated from wild-type, TLR-2^–/–, and TLR-4^–/– mice to exhibit markers of cell death in response to C. pneumoniae infection was evaluated. As shown in Fig. 6, the absence of TLR-2 but not the absence of TLR-4 ablated the induction of annexin V binding in response to C. pneumoniae following 24 h of treatment.

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FIG. 6. The absence of TLR-2 but not the absence of TLR-4 ablates the inductive effects of oxidized LDL and C. pneumoniae infection on binding of annexin V by bone marrow macrophages. Bone marrow macrophages were isolated from wild-type C57BL/6 mice (WT) or from mice devoid of Toll-like receptor 2 (TLR 2–/–) or Toll-like receptor 4 (TLR 4–/–) with a C57BL/6 background. The cells were either not treated, incubated for 24 h with 5 µg/ml oxidized LDL, or infected for 24 h with live C. pneumoniae at an MOI of 0.01. The binding of fluorescein-labeled annexin V was measured by flow cytometry. An asterisk indicates that the P value is <0.05 for a comparison with no-treatment control cells. A dagger indicates that the P value is <0.05 for a comparison with wild-type or TLR-4–/– cells either treated with oxidized LDL or infected with C. pneumoniae. The data are the means ± standard deviations of triplicate determinations.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The results of this study show that C. pneumoniae infection has an additive effect with oxidized LDL on the induction of markers of cell death in macrophages. There are a variety of mechanisms by which oxidized lipids and free cholesterol have been reported to kill macrophages. For example, recent evidence suggests that oxidized LDL induces apoptosis both through activation of death receptors and through mitochondrial pathways. It has also recently been reported that the oxysterols, lipid hydroperoxides, oxidized phospholipids, and free cholesterol found within oxidized LDL independently induce death (2, 5, 11, 27, 28, 30, 36, 47, 50, 51, 53-55). In the present study and in keeping with previous observations, it was found that even very low concentrations of oxidized LDL were capable of inducing markers of apoptosis in the RAW cells, including mitochondrial pore formation, activation of caspase 3, and fragmentation of DNA.

C. pneumoniae is an obligate intracellular pathogen, and as such this organism is dependent on the continued functioning of the host cell to support bacterial growth and the capacity for sustained infection. Thus, it is logical to assume that C. pneumoniae developed some means for preventing the host cell from undergoing apoptosis. However, there have been varied reports that chlamydiae and other obligate intracellular bacteria both inhibit and induce host cell death prior to completion of their growth cycles. Several groups have reported that infection of epithelial cells by C. pneumoniae, C. trachomatis, and C. psittaci inhibits apoptosis induced by staurosporin or engagement of death receptors. This inhibitory effect appears to be mediated by stimulation of NFB activation, inhibition of cytochrome c release from mitochondria, and the subsequent inhibition of caspase activation (17, 40, 48, 49). However, another recent study demonstrated that C. pneumoniae inhibition of apoptosis is not due to NFB-dependent factors (13).

In contrast, other groups have reported that C. pneumoniae, C. trachomatis, and C. psittaci stimulate cell death in epithelial cells, macrophages, and other cell types (16, 35, 37-39, 43). Furthermore, other types of obligate intracellular gram-negative bacteria, such as Legionella longbeachae, have been shown to induce apoptosis in macrophages (34).

Although it is currently not known precisely how chlamydiae induce death, it has recently been reported that C. trachomatis encodes a protein now termed "chlamydia protein associating with death domains" (CADD) that activates death receptors and induces apoptosis in a variety of mammalian cell lines. C. pneumoniae and C. muridarum contain open reading frames encoding hypothetical proteins exhibiting sequence similarity and predicted structural homology to CADD (44, 45).

C. pneumoniae was observed to induce markers of cell death within 24 h after exposure to the organism. Paradoxically, C. pneumoniae infection of macrophages is productive, although the burst size is smaller than that observed for other cell types (15). This suggests that organisms can survive in host cells undergoing these processes but that the lower burst size may reflect decreased growth or multiplication due to a stressed host environment.

The observation that C. pneumoniae stimulates annexin V binding and PI uptake within 24 h but does not activate caspase 3 or induce mitochondrial pore formation or fragmentation of DNA suggests that the C. pneumoniae-induced death of macrophages occurs through a "necrotic" mechanism. Necrosis is defined as cell death in the absence of apoptotic responses that cannot be inhibited by BCL-2 or by inhibition of caspases (23, 25). Typically, annexin V binding without uptake of PI is a hallmark of the early stages of apoptosis (31) and may even play a physiologic role in stimulation of coagulation (16). The uptake of PI, on the other hand, is indicative of a loss of membrane integrity and accompanies the later stages of apoptotic death. It is, however, an early and predominant effect of necrotic death (25). This type of necrotic death has previously been reported in RAW cells infected with both live and heat-inactivated Escherichia coli (23). Furthermore, rapid and transient externalization of phosphatidylserine has been reported for epithelial cells, endothelial cells, and granulocytic and monocytic cell types that were infected with C. pneumoniae or C. trachomatis, and the response was not blocked with a caspase inhibitor (16). However, in a recent study, C. pneumoniae infection was shown to cause smooth muscle cell death through a mixture of apoptotic features (DNA fragmentation, externalization of phosphatidylserine, and loss of cytochrome c staining) and necrotic features (early membrane damage of plasma membrane and organelles), leading to the conclusion that C. pneumoniae induced aponecrosis in smooth muscle cells (10).

The death of the host cell through a necrotic pathway with accompanying loss of membrane integrity may involve rapid increases in intracellular calcium levels that in turn activate calcium-dependent calpains. Calpains, like caspases, can cleave critical cellular proteins (25). In fact, C. pneumoniae has been shown to activate an L-type calcium channel in macrophages, leading to an increased influx of calcium, and this has been attributed to chlamydial LPS, suggesting that this process is potentially mediated by Toll-like receptors (3). Interestingly, ox-LDL also alters the transmembrane calcium gradient in macrophages (52). However, it is currently not clear how C. pneumoniae specifically induces loss of membrane integrity and necrotic death. One possibility is signaling through Toll-like receptors. It is well known that both TLR-2 and TLR-4 ligands induce caspase-dependent apoptosis in cells in which protein synthesis is blocked or in which NFkB activation is prevented (19, 21, 22, 26, 32, 42, 56). However, it also appears that Toll-like receptor engagement may induce a necrotic form of cell death as well. Kirschnek et al. (23) recently reported that treatment of RAW cells with the TLR-4 ligand LPS induces a modest increase in the uptake of PI without blocking protein synthesis and that this occurs prior to induction of apoptosis. They also showed that the combination of LPS plus the TLR-2 ligand Pam3Cys and the TLR-9 ligand CpG oligonucleotide greatly increased the uptake of PI and that the increase in necrosis was not mediated by the Toll-like receptor-induced secretion of tumor necrosis factor alpha. The current data showing that the absence of TLR-2 but not TLR-4 in bone marrow-derived macrophages inhibits C. pneumoniae-induced binding of annexin V and that inactivated forms of C. pneumoniae stimulate annexin V binding and the uptake of PI within 24 h of treatment without stimulating apoptosis are consistent with the observations of Kirschnek et al. (23). Whether the necrotic death involves increased intracellular calcium and the activation of calpains is currently being investigated.

In summary, the results of this study showed that both live and inactivated forms of C. pneumoniae induce a necrotic form of cell death which augments the apoptotic cell death induced by the accumulation of oxidized LDL by macrophages. As the death of foam cells is fundamental to the progression and destabilization of atherosclerotic lesions, these observations suggest that C. pneumoniae infection may contribute to destabilization of atherosclerotic plaques and may help explain why C. pneumoniae is now considered to be an independent risk factor for cardiovascular disease.

 
This study was supported by Public Health Service grants R01HL66115 (to M.E.R.), R01 HL62995 (to W.C.L.), and R01 HL56036 (to C.-C.K.) from the National Heart, Lung, and Blood Institute.

We thank Amy Lee and Jerry Ricks for their excellent technical assistance and Thomas Q. Nhan for input into the design and interpretation of this study.

 
* Corresponding author. Mailing address: Department of Pathobiology, Box 353410, University of Washington, Seattle, WA 98195. Phone: (206) 543-1738. Fax: (206) 616-1245. E-mail: ssmjm{at}u.washington.edu.

Editor: J. N. Weiser

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Infection and Immunity, July 2005, p. 4315-4322, Vol. 73, No. 7
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.7.4315-4322.2005
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