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Cellular Oxidation of Low-Density Lipoprotein by Chlamydia pneumoniae

  1. Murat V. Kalayoglu1,
  2. Brian Hoerneman1,
  3. David LaVerda1,
  4. Sandra G. Morrison2,
  5. Richard P. Morrison2 and
  6. Gerald I. Byrne1
  1. Department of Medical Microbiology and Immunology, University of Wisconsin Medical School, Madison
  2. Department of Microbiology, Montana State University, Bozeman
  1. Reprints or correspondence: Dr. Gerald I. Byrne, Dept. of Medical Microbiology and Immunology, University of Wisconsin Medical School, 471 SMI, 1300 University Ave., Madison, WI53706 (gibyrne{at}facstaff.wisc.edu).
 
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Abstract

A spectrum of clinical and epidemiologic studies implicate infectious agents, including Chlamydia pneumoniae, in the pathogenesis of atherosclerosis. The complexity of atherosclerotic disease necessitates examining the role of infection in the context of defined risk factors, such as high levels of native low-density lipoprotein (LDL). Although native LDL does not have atherogenic properties, cellular oxidation of LDL alters the lipoprotein into a highly atherogenic form. In this report, C. pneumoniae and chlamydial hsp60, an inflammatory antigen that was recently localized to atheromas, were found to induce cellular oxidation of LDL. These data provide initial evidence that an infectious agent can render LDL atherogenic and suggest a mechanism whereby C. pneumoniae may promote atheroma development.

Atherosclerosis and its complications lead to half of all adult deaths in the United States [1, 2]. The lesion, or atheroma, is an inflammatory site where a variety of cells, cell products, and lipoproteins interact to promote injury and disease [3]. An important consequence of these interactions is the cellular oxidation of low-density lipoprotein (LDL), which alters the lipoprotein to a highly atherogenic form [4, 5]. A variety of cell types present in atherosclerotic lesions, including monocytes/macrophages, smooth muscle cells, and endothelial cells, can oxidize LDL [5]. In turn, oxidized LDL promotes cell injury, smooth muscle cell proliferation, foam cell formation, chemotaxis of leukocytes, cellular secretion of inflammatory mediators, and other events that modulate atheroma biology [6–9]. Oxidized LDL has been detected in atheromas of rabbits and humans, and antioxidant therapy may decrease cardiovascular events and mortality [10, 11], perhaps by inhibiting the oxidation of lipoproteins. Therefore, oxidation of LDL, a biologically plausible mechanism of LDL modification, may explain why high plasma levels of native LDL are a major risk factor for coronary artery disease.

Appreciation of atherosclerosis as an inflammatory disease has fostered interest in the role that infectious agents may play in atheroma development [12]. In particular, the intracellular prokaryotic pathogen Chlamydia pneumoniae has been associated with atherosclerosis by an array of epidemiologic and clinical studies [13]. Initial work by Saikku et al. [14] demonstrated higher titers of anti—C. pneumoniae antibodies in patients with atherosclerosis than in healthy controls. These findings have now been confirmed in 19 separate studies [12]. Furthermore, C. pneumoniae was associated with atherosclerosis by polymerase chain reaction (PCR), which showed the organism's DNA within atheromas, and by localization of the pathogen to atheromas by electron microscopy and immunohistochemistry [15–17]. The organism has also been isolated and propagated from atheromas [18]. Animal models show that C. pneumoniae infection initiates vascular atheromatous changes in rabbits [19, 20] and promotes atheroma development in apolipoprotein E knockout mice [21]. Antibiotic treatment of infected rabbits inhibits these atheromatous changes [22]. Moreover, antichlamydial antibiotics reduce cardiovascular events in persons with coronary artery disease [23, 24]. Although these data establish a strong association between the pathogen and atherosclerosis, little is known about how the organism contributes to lesion development. To explore one pathogenic mechanism of disease, we examined the capacity of C. pneumoniae to induce LDL oxidation by human monocytes.

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Materials and Methods

Reagents

Unless otherwise noted, all reagents were purchased from Sigma (St. Louis).

Preparation of LDL

LDL was isolated from normolipidemic donors by density-gradient ultracentrifugation as described elsewhere [25], extensively dialyzed against 0.15 M NaCl and 0.05% EDTA, and concentrated by centrifugation with Centri/Por concentrators (Spectrum Medical Industries, Houston). LDL protein and cholesterol were determined by a protein assay (Bio-Rad Laboratories, Richmond, CA) and a total cholesterol test kit (Sigma), respectively. SDS-PAGE analysis of LDL on a 4%–20% gradient gel revealed a single protein (MW ∼250,000) consistent with LDL apolipoprotein B-100. Agarose gel electrophoresis of LDL showed no increase in relative electrophoretic mobility within 2 weeks, indicating that isolated LDL did not become oxidized. Isolated LDL was stored at 4°C and used within 2 weeks of isolation. LDL concentrations are reported as micrograms of protein per milliliter.

Propagation and storage of C. pneumoniae

C. pneumoniae (TW-183) was purchased (American Type Culture Collection, [ATCC] Rockville, MD), propagated in HEp-2 cells (ATCC), and purified by renografin-gradient centrifugation, as described elsewhere [26]. All Chlamydia stocks and cell lines were consistently negative for Mycoplasma DNA by commercial polymerase chain reaction—ELISA kit (Boehringer Mannheim, Indianapolis). Propagated organisms were stored at −70°C in sucrose buffer and titered for infectivity [27]. Titers are reported here as infection-forming units (ifu), where 1 ifu/cell equals 1 infectious Chlamydia/HEp-2 cell. To eliminate interference from sucrose buffer in oxidation assays, chlamydiae were thawed, washed twice in endotoxin-free sterile water, and resuspended in 100 µL of water before being added to monocytes. In some experiments, C. pneumoniae was exposed to heat for 30 min at 95°C or UV irradiation for 30 min at 15.25 cm from a 30-W germicidal light source (OSRAM Sylvania, Danvers, MA). In separate experiments, these treatments were found to inhibit infectivity 11000-fold by titration on HEp-2 cell monolayers.

Preparation of heat-shock proteins (hsp)

The C. trachomatis homologue of hsp60 (chsp60) was purified from recombinant clone JM109 (pTA571) by immunoaffinity columns using antichlamydial hsp60 monoclonal antibody (MAb) A57-B9 as described [28]. chsp10 containing a C-terminal His6 tag was purified by nickel chelate affinity chromatography as described elsewhere [29]. SDS-PAGE analysis yielded a single band migrating at ∼60 kDa (chsp60) and 12 kDa (chsp10). Preparations were not contaminated with lipopolysaccharide (LPS) by the limulus assay (Biowhittaker, Walk-ersville, MA). Sensitivity of the assay was 6 pg/mL LPS.

Preparation of opsonized zymosan (ZOP)

Zymosan (Sigma) was opsonized by the method of Johnston [30]. In brief, 10 mg/mL zymosan was suspended in phosphate-buffered saline (PBS), boiled for 30 min, washed twice with PBS, and incubated at a 1: 4 dilution with fresh human serum for 30 min at 37°C. The preparation then was washed and resuspended in sterile PBS to use in cell culture assays.

Isolation of human monocytes and culture conditions

Human mononuclear cells were isolated by ficoll-hypaque centrifugation from blood from healthy donors. We added 106 mononuclear cells to 96-well microtiter plates (0.32 cm2 growth area; Costar, Cambridge, MA), incubated the plates at 37°C for 30–45 min in RPMI 1640 medium (Biowhittaker), and washed them 6 times with Hanks' balanced salt solution. Adherent cells (∼1–2 × 105 cells/well) were 190% monocytes, as determined by anti-CD 14 MAb staining (Sigma) and were 195% viable by trypan blue staining. Varying concentrations of LDL and C. pneumoniae were added to monocytes or empty wells, and cultures were incubated for 18–40 h. Separate studies showed that monocytes are infected efficiently under these conditions by incubating cells with C. pneumoniae for 3–4 days, fixing and staining cells with MAb specific for chlamydial lipopolysaccharide (LPS), and counting inclusions (not shown). All assays were done in RPMI 1640 medium, which does not contain transitional metal ions. The medium was not supplemented with fetal calf or human serum, as the high concentration of antioxidants in serum completely abrogate LDL oxidation [5]; indeed, the inclusion of 5% serum completely inhibited C. pneumoniae—induced cellular LDL oxidation (MV..K., G.I.B., unpublished data). In experiments involving vitamin E, the reagent was added to LDL either before or concurrent with addition of the cells; there was no significant difference in inhibition of oxidation by either method.

Thiobarbituric acid (TBA)-reactive substances assay (TBARS)

LDL oxidation was measured by quantitating TBARS by fluorometry, as described elsewhere [31]. TBARS take advantage of the capacity of aldehydes, which form during LDL oxidation, to combine with TBA to yield a fluorochrome. This assay has been modified in our laboratory to detect small quantities of TBARS using monocytes plated on microtiter plates. Supernatants were collected at various times after treatment and clarified by centrifugation, and protein was precipitated by treatment with 25% trichloroacetic acid and centrifuged again. Supernatants then were mixed with an equal volume of 1% TBA, heated at 95°C for 45 min, cooled, and read by fluorometry at 520 nm excitation, 550 nm emission. Concentrations of LDL oxidation products were determined from a standard curve constructed using malondialdehyde (MDA), which reacts with TBA to yield fluorometric MDA-TBA adducts. Oxidation values are expressed as nanomole of MDA per milliliter. Data are reported as means of triplicate experiments; SDs are representative of ⩾3 experiments.

Relative electrophoretic mobility (REM) shift assay

In some experiments, LDL oxidation was measured by agarose gel electrophoresis to detect an increase in electrophoretic mobility of oxidized LDL relative to native LDL [32]. In brief, 10 µL of supernatant clarified by centrifugation was loaded into a 0.5% agarose gel and electrophoresed using tricine buffer (pH 8.6; Bio-Rad) for 90 min at 100 V constant voltage. The gel was fixed in a solution containing 5% acetic acid and 75% ethanol for 15 min, stained with 1% oil red O (in 60% isopropanol) for 30 min, and rinsed with distilled water to visualize LDL bands. Migration of each LDL band was measured in millimeters and is expressed here as REM compared with a native LDL control. REM values are representative of ⩾3 experiments.

Fibroblast cytotoxicity assay

Human foreskin fibroblasts immortalized by retrovirus-mediated gene transfer of the E6 and E7 genes of human papillomavirus type 16 [33] were a gift of T. Compton (Madison, WI). Fibroblasts were maintained in Dulbecco's modified Eagle medium supplemented with 5% fetal bovine serum, L-glutamine, and antibiotics, and plated at a density of 104 cells/well in 96-well microtiter plates (Costar) overnight before cytotoxicity assays. Cytotoxicity was assessed essentially as described [34]. In brief, cells were washed 3 times with Dulbecco's PBS (D-PBS) and incubated with 50 µL of conditioned supernatant for 36 h. Fibroblasts were washed 5 times with D-PBS, and ∼500 cells/well were counted at ×200 magnification. Cell counts are expressed as percent of mock-infected controls. In some experiments, viability was assessed by staining with 10 µg/mL calcine AM (Molecular Probes, Eugene, OR) and counting fluorescent cells at ×200. No cytotoxicity was observed in fibroblasts cultured with supernatant from mock-infected, LDL-treated, or C. pneumoniae—exposed, non-LDL-treated monocytes. Data are reported as means of triplicate experiments; SDs are representative of ⩾3 experiments.

Superoxide anion release

Superoxide release by human monocytes was quantitated by measuring the superoxide dismutase (SOD)-inhibitable reduction of cytochrome c [35], which is reduced on a mole-for-mole basis by superoxide, leading to an increased absorbance at 550 nm. In brief, we incubated treated monocytes for varying times in PBS supplemented with 2 mg/mL dextrose and 80 mM ferricytochrome c (Sigma; from horse heart) in the presence or absence of 300 U/mL SOD and determined the absorbance of supernatants at 550 nm. The production of SOD-inhibitable superoxide was calculated by determining the absorbance of supernatants at 550 nm and then subtracting the respective SOD controls from each sample. Data are presented as SOD-corrected single values and are representative of 3 experiments.

Results

C. pneumoniae induces cellular oxidation of LDL

C. pneumoniae—exposed and mock-infected monocytes were cultured in the presence of LDL, and oxidation was quantitated by measuring TBARS (figure 1A), a shift in electrophoretic mobility of oxidized LDL relative to native LDL (figure 1B), and cytotoxicity of oxidized LDL to human fibroblasts (figure 1C), a feature consistent with pathologic changes in atheroma development. Data obtained by each method showed that C. pneumoniae exposure caused monocytes to oxidize LDL in a dose-dependent manner (P < .05 for 2, 4, and 8 ifu/mL of C. pneumoniae-exposed monocytes, compared with mock-infected samples). LDL-free controls consistently had minimal oxidation products (<0.5 nM MDA/mL), indicating that the TBARS were produced only in the presence of LDL. The production of TBARS by C. pneumoniae-exposed monocytes (figure 1A) correlated directly with a shift in the REM of oxidized LDL (figure 1B) and with cytotoxicity of oxidized LDL to fibroblasts (figure 1C). Supernatants from monocytes cultured in the presence of 2.5 IU/mL of vitamin E or in the absence of LDL were not cytotoxic to fibroblasts, indicating that cytotoxicity occurred as a result of oxidized LDL. Furthermore, exposure of fibroblasts to C. pneumoniae alone did not result in cytotoxicity. To determine the kinetics of C. pneumoniae-induced cellular oxidation of LDL, C. pneumoniae-exposed monocytes were cultured in the presence of LDL for ⩽40 h. A linear increase in the production of TBARS was observed throughout the course of the experiment (see figure 2A; P < .05 for C. pneumoniae—exposed monocytes incubated for 18–40 h vs. 0 h) and paralleled an increase in REM (not shown).

Figure 1
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Figure 1

Oxidation of low-density lipoprotein (LDL) by monocytes exposed to Chlamydia pneumoniae. Varying concentrations of C. pneumoniae (or equivalent volumes) were incubated with (circles) or without (triangles) monocytes in presence (open symbols) or absence (filled symbols) of 400 µg/mL LDL for 18 h and assayed for production of (A) thiobarbituric acid—reactive substances (TBARS) or (B) shift in electrophoretic mobility relative to native LDL. Relative electrophoretic mobility (REM) shift assay measured migration of single bands from origin. Data are representative of similar findings. (C) In cytotoxicity assays, monocytes were incubated with 400 µg/mL LDL with varying concentrations of C. pneumoniae for 24 h; supernatant was clarified by centrifugation, and monolayers of human fibroblasts were incubated with conditioned supernatants for 36 h. Viable fibroblasts remaining after 36 h (expressed as % fibroblasts that received supernatants from mock-infected monocytes) are plotted as function of C. pneumoniae dose (●). Supernatants also were assayed for TBARS production before addition to fibroblasts (○). Fibroblasts exposed directly to C. pneumoniae and cultured in presence or absence of LDL did not exhibit cytotoxicity. SDs (where error bars not visible), <0.181 nM malondialdehyde (MDA)/mL (TBARS assay) or 2.4% (cytotoxicity assay); IFU, infection-forming units.

Figure 2
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Figure 2

Characterization of Chlamydia pneumoniae—induced monocyte oxidation of low-density lipoprotein (LDL). A, Monocytes cultured in presence (○) or absence (●) of C. pneumoniae (3 infection-forming units [ifu]/cell) and 100 µg/mL LDL for indicated times and assayed for production of thiobarbituric acid-reactive substances (TBARS). B, C. pneumo niae-exposed (4 ifu/cell, ○) and mock-infected (●) monocytes were cultured with varying concentrations of LDL for 18 h before assay for TBARS production. C, C. pneumoniae—exposed (○) and mock-infected (●) monocytes incubated with 400 µg/mL LDL and varying concentrations of vitamin E (α-tocopherol) for 18 h and assayed for TBARS. Oxidation values were corrected by subtracting from cell-free controls. SDs (where error bars not visible), <0.181 nM malondialdehyde (MDA)/mL.

To examine whether high LDL concentrations, an established risk factor for atherosclerosis [36], enhanced C. pneumoniae—induced cellular LDL oxidation, C. pneumoniae—exposed and mock-infected monocytes were incubated in the presence of specified amounts of LDL. More oxidation was observed at higher concentrations of LDL (figure 2B; P < .05 for C. pneumoniae—exposed monocytes incubated with >100 µg/mL LDL, compared with LDL-free controls). Furthermore, the antioxidant vitamin E (α-tocopherol) inhibited C. pneumoniae—induced monocyte oxidation of LDL in a dose-dependent manner (figure 2C). We found that 2.5 IU/mL of vitamin E was sufficient to completely inhibit LDL oxidation (P < .05 vs. untreated C. pneumoniae—exposed samples). Collectively, these data show that an infectious agent can induce cellular oxidation of LDL and suggest a pathogenic mechanism for C. pneumoniae in atherosclerosis.

C. pneumoniae—induced cellular LDL oxidation occurs by a superoxide-independent mechanism. Activated monocyte-mediated oxidation of LDL is thought to occur by the release of superoxide anions, which directly oxidize LDL [37–39] and can reduce transition metal ions that subsequently facilitate lipid hydroperoxide decomposition and chain peroxidation [40]. To determine whether C. pneumoniae—induced monocyte oxidation of LDL involved the production of superoxide, the release of superoxide anions by C. pneumoniae—exposed monocytes was monitored for the first 80 min after treatment. C. pneumoniae did not cause increased production of superoxide anions by monocytes (figure 3A). In contrast, monocytes treated with ZOP, a preparation commonly used to study the cellular oxidation of LDL, released superoxide anions in a time-dependent manner. To test whether the degree of cellular superoxide production correlated with LDL oxidation, the C. pneumoniae—exposed or zymosan-treated monocytes were cultured in the absence or presence of LDL and tested for the production of TBARS after 24 h (figure 3B). C. pneumoniae induced a high degree of specific cellular LDL oxidation despite an incapacity to activate the monocyte respiratory burst. Zymosan induced a smaller degree of LDL oxidation (figure 3B), which was completely inhibited by 50–120 U/mL of SOD in separate experiments (data not shown). In contrast, SOD had a minimal inhibitory effect on C. pneumoniae—induced cellular LDL oxidation (figure 3C). Catalase (100 U/mL) also minimally inhibited C. pneumoniae—induced cellular LDL oxidation (data not shown). Thus, the capacity of this pathogen to induce monocyte oxidation of LDL does not seem to depend on the generation of superoxide by C. pneumoniae-exposed monocytes.

Figure 3
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Figure 3

Chlamydia pneumoniae—induced cellular low-density lipoprotein (LDL) oxidation occurs by superoxide-independent mechanism. A, Monocytes were untreated (●), treated with 100 ng/mL interferon (IFN)—g + 5 × 10−6 M PMA (○, positive control), 1 µg/mL opsonized zymosan (ZOP; △), or 1.2 infection-forming units (ifu)/cell C. pneumoniae (▴), incubated for varying times, and assayed for superoxide production as described in Methods. B, Monocyte replicates plated from A were cultured in presence (open bars) or absence (filled bars) of 100 µg/mL LDL and assayed for thiobarbituric acid-reactive substance (TBARS) production. C, C. pneumoniae-exposed (4 ifu/cell; O) or mock-infected (●) monocytes incubated with 100 µg/mL LDL and varying concentrations of superoxide dismutase (SOD) for 24 h and assayed for TBARS production. Where error bars not visible (TBARS assay), SDs are <0.181 nM malondialdehyde (MDA)/mL.

chsp60 can induce cellular LDL oxidation

To determine the nature of chlamydial antigens necessary to induce cellular LDL oxidation, chlamydiae were inactivated with either heat or UV light, added to monocytes in the presence of LDL, and assayed for LDL oxidation products. Treatment of chlamydiae with heat nearly abolished the capacity of C. pneumoniae to induce LDL oxidation (P < .05, compared with C. pneumoniae—exposed samples), whereas UV light had only a minimal effect (figure 4A). Thus, heat-labile chlamydial components appear to contribute to the induction of LDL oxidation. The heat-stable purified chlamydial LPS induced cellular oxidation only at very high concentrations (120 µg/mL). Furthermore, addition of the LPS antagonist polymyxin B or MAb (MY4 clone) to the CD14 LPS receptor did not inhibit C. pneumoniae—induced cellular oxidation (not shown). However, purified chsp60, an inflammatory protein associated with chronic chlamydial infections [41] and recently localized to atheroma macrophages [42], induced a dose-dependent increase in cellular LDL oxidation as measured by the production of TBARS (figure 4B), a shift in REM (figure 4C), and cytotoxicity of conditioned supernatant to human fibroblasts (figure 4D; P <.05 for 0.172, 0.516, and 1.548 nM/mL chsp60-treated monocytes vs. untreated samples). In addition, when chsp60 was treated with heat, cellular oxidation of LDL decreased by 82%, also similar to C. pneumoniae heat treatment. The cytotoxicity assay showed an inverse correlation of viable fibroblasts to production of TBARS (figure 4D). chsp60 alone was not cytotoxic to fibroblasts. Furthermore, supernatants from monocytes cultured in the presence of vitamin E or in the absence of LDL exhibited no cytotoxicity to fibroblasts, indicating that cytotoxicity was specific to oxidized LDL. As expected, vitamin E also inhibited TBARS production (figure 5A; P <.05 vs. untreated samples). Another recently cloned and purified chsp (chsp10) did not induce LDL oxidation (figure 5B; P > .05 for all concentrations of chsp10 vs. untreated samples). In sum, these data identify chsp60 as a chlamydial component capable of inducing cellular oxidation of LDL.

Figure 4
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Figure 4

Demonstration of chlamydia hsp60 (chsp60) as major chlamydial component that promotes cellular oxidation of low-density lipoprotein (LDL). A, Monocytes were cultured for 18 h with 400 µg/mL LDL in presence or absence of Chlamydia pneumoniae that was untreated or treated with heat or UV radiation. Oxidation was measured by quantitating thiobarbituric acid-reactive substances (TBARS) production. B, Varying concentrations of purified chsp60 were incubated with (circles) or without (triangles) monocytes in presence (open symbols) or absence (filled symbols) of 400 µg/mL LDL for 18 h and assayed for TBARS production [16]. C, chsp60 was incubated as in B and assayed for shift in electrophoretic mobility relative to native LDL. Relative electrophoretic mobility (REM) shift assay measured migration of single bands from origin and is representative of similar findings. D, In cytotoxicity assays, monocytes and 400 µg/mL LDL were incubated with varying concentrations of chsp60 for 24 h, supernatant was clarified by centrifugation, and monolayers of human fibroblasts were incubated with conditioned supernatants for 36 h. Viable fibroblasts remaining after 36 h (expressed as % fibroblasts that received supernatants from mock-infected monocytes) are plotted as function of C. pneumoniae dose (●). Supernatants also were assayed for TBARS production before addition to fibroblasts (○). Fibroblasts exposed directly to chsp60 and cultured with or without LDL did not exhibit cytotoxicity. Where error bars not visible, SDs are <0.181 nM malondialdehyde (MDA)/mL (TBARS assay) or 2.4% (cytotoxicity assay).

Figure 5
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Figure 5

Characterization of cellular low-density lipoprotein (LDL) oxidation by chlamydial heat-shock proteins (cshp). Oxidation values were subtracted from cell-free controls and expressed as nM malondialdehyde (MDA)/mL. A, Monocytes were incubated in presence of 400 µg/mL LDL with (hatched bars) or without (closed bars) 0.765 nM/mL chsp60 and with or without 2 IU/mL vitamin E for 18 h and assayed for production of thiobarbituric acid-reactive substances (TBARS). B, Oxidation of LDL by monocytes treated with chsp60 (○) or hsp10 (●). Monocytes were incubated with 400 µg/mL LDL and indicated concentrations of chsp for 18 h and assayed for TBARS production. Where error bars not visible, SDs are <0.181 nM MDA/mL.

Discussion

Data from clinical studies implicate a role for infectious agents in the pathogenesis of atherosclerosis [12]. Because atherosclerosis is a complex disease process, the role of any pathogen in atheroma development must be considered in the context of other risk factors. An established risk factor in atherosclerosis is high levels of serum LDL cholesterol [36]; however, in its native form, LDL does not cause changes associated with atheroma initiation or progression [32].

One hypothesis is that infectious agents serve as a link between high serum LDL levels and an event critical to the development of the atheroma, such as the cellular oxidation of LDL. Oxidized LDL displays multiple atherogenic properties, including dysregulation of vascular tone, injury to the endothelium, promotion of leukocyte entry into the vessel wall, smooth muscle cell migration and proliferation, and foam cell formation [6–9, 43, 44]. Although serum antioxidants protect native LDL from oxidation in the macrovasculature, the atherosclerotic lesion contains oxidized LDL and may permit oxidation by acting as a sequestered microenvironment [5]. The primary aim of the current study was to determine whether cellular LDL oxidation can be enhanced by C. pneumoniae and, if so, what chlamydial components may be responsible for this event. Our data show that an infectious agent can induce cellular oxidation of LDL and suggest that C. pneumoniae can serve as a link between a known risk factor and an important atherogenic event.

A small amount of LDL oxidation was detected when C. pneumoniae (or chsp60) was cultured with LDL in the absence of cells (figure 1A, △), which may be due to direct interaction between C. pneumoniae (or chsp60) and LDL or the presence of any residual transitional elements on chlamydial elementary bodies. This control also showed that C. pneumoniae induced a significant increase in cellular LDL oxidation independent of the direct interaction between C. pneumoniae and LDL (compare figure 1A, ○ vs. △). It is of interest that C. pneumoniae did not promote the release of superoxide by monocytes (figure 3), suggesting that superoxide-independent mechanisms were involved in C. pneumoniae—induced monocyte oxidation of LDL. This was unexpected, because previous studies [45—47] showed that monocytes activated with high concentrations (∼2 µg/mL) of ZOP oxidize LDL by superoxide-dependent mechanisms; in fact, in our hands, ZOP also induced SOD-inhibitable monocyte oxidation of LDL (see Results). However, induction of superoxide may not be necessary to induce LDL oxidation by macrophages [48], and superoxide alone may not directly oxidize LDL by monocytes [49]. Indeed, chlamydiae use a poorly understood, unique entry mechanism that does not resemble ZOP-induced phagocytosis. Furthermore, recent work suggests that ZOP may not be a physiologically relevant agent to activate monocytes for study of LDL oxidation, because it is contaminated with iron in many commercial preparations [49]. Such differences in particle entry, size, and composition between chlamydiae and ZOP probably lead to independent pathways of macrophage activation. The inability of this organism to promote superoxide release by monocytes is not surprising, given that other chlamydial species also do not induce the phagocyte respiratory burst [50, 51]. Although the mechanism by which Chlamydia inhibit the respiratory burst remains unclear, such inhibition clearly favors survival for an obligate intracellular pathogen. Of interest, chlamydiae can induce a selective release of myeloperoxidase without promoting superoxide production [52]. This observation may be relevant to C. pneumoniae—induced monocyte activation, as myeloperoxidase has been proposed to play an important role in the oxidation of LDL in vivo [53].

Other potential mechanisms for C. pneumoniae—mediated macrophage LDL oxidation include induction of lipoxygenase [54], NADPH oxidase [55], thiol recycling [56], and transitional metal ions [57]. Monocyte-mediated LDL oxidation also may involve an increase in intracellular calcium and the activation of protein kinase C [47]. Ongoing work will establish the precise mechanism of oxidation; the use of serum-free RPMI medium, which does not contain transitional metal ions, suggests that C. pneumoniae—induced cellular LDL oxidation does not depend on the reduction of copper or iron.

Atherosclerosis and chlamydial diseases are both chronic inflammatory conditions that may result from a variety of risk factors [3, 41], and clinical studies suggest that chronic, persistent chlamydial infections occur in vivo [58]. A noninfectious but viable persistent form of C. pneumoniae [59] and C. trachomatis [60] can be induced under a variety of conditions, most notably following treatment with interferon-γ, an activated T cell cytokine detected in atheroma [61]. In this metabolically quiescent state, persistent chlamydiae express minimal levels of the immunoprotective major outer membrane protein but high levels of the immunopathologic antigen chsp60 [58, 60, 62]. chsp60, recently localized within atheroma macrophages [42], is strongly associated with a spectrum of chronic sequelae of C. trachomatis infection, including trachoma, reactive arthritis, and pelvic inflammatory disease [41]. The observation that chsp60 can induce cellular oxidation of LDL identifies a chlamydial component that may promote an important atherogenic event and suggests that this antigen also plays a key role in atheroma development. However, in the absence of any agents that specifically block chsp60 function, we cannot rule out the possibility that chlamydial components in addition to chsp60 also may contribute to the oxidation process.

In addition to inducing cellular LDL oxidation, chsp60 may promote atherogenesis by other mechanisms. Kol et al. [63] recently showed that chsp60 can activate human vascular endothelium, smooth muscle cells, and macrophages, and Mayr et al. [64] demonstrated endothelial cytotoxicity mediated by serum antibodies to chsp60. To this end, it is significant that stress response proteins may exhibit disease-provoking mechanisms that extend beyond their well-documented role as antigens. Retzlaff et al. [65] found that hsp60 from Legionella pneumophila directly induced interleukin-1β mRNA via protein kinase C signaling. This observation is of interest in that it provides evidence that host cells are equipped with hsp60-recognizing receptors to initiate signal transduction systems. Surface receptors to hsp60 were further substantiated by Garduno et al. [66], who showed that hsp60 is surface exposed on L. pneumophila and mediates invasion into HeLa cells via specific receptor-ligand interactions [67]. Importantly, the observations by Kol et al. [42], which showed that chsp60 directly stimulates induction of macrophage matrix metalloproteinase, suggest a direct signaling role for chsp60 in atherogenesis.

We recently showed that C. pneumoniae interacts with monocyte-derived macrophages to dysregulate lipoprotein uptake and induce macrophage foam cell formation [27], the hallmark of initial lesions in atherosclerosis. In the current report, C. pneumoniae induced monocyte oxidation of LDL, a key event in atheroma development. Modulation of mononuclear phagocyte functions by C. pneumoniae therefore may lead to atherogenic alterations in lipoprotein metabolism within initial fatty streaks and subsequently progressing atheroma. We believe that foam cell formation occurs independently of oxidation, in part because serum and other antioxidants, such as vitamin E, inhibit C. pneumoniae—induced macrophage LDL oxidation but not foam cell formation. Furthermore, while induction of lipid ingestion is a relatively early event (within 2 h of stimulation) [68], appreciable LDL oxidation does not occur until ∼18 h after exposure. Importantly, distinct C. pneumoniae components may be responsible for macrophage foam cell formation and monocyte oxidation of LDL; the principal C. pneumoniae component that induced macrophage native lipoprotein uptake and foam cell formation was chlamydial LPS [69], whereas this report shows that primarily heat-labile components of C. pneumoniae, such as chsp60, induce monocyte LDL oxidation. Coupling these and other chlamydial components to defined atherogenic functions will help elucidate pathogenic mechanisms and may serve to establish a causal role for C. pneumoniae in atherosclerosis.

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Acknowledgments

We thank Charles Schobert and Adam Pleister for helping to propagate C. pneumoniae and Teresa Compton for providing immortalized human fibroblasts.

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Footnotes

  • Grant support: NIH (AI-42790).

  • Received November 2, 1998.
  • Revision received April 9, 1999.
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  1. J Infect Dis. (1999) 180 (3): 780-790. doi: 10.1086/314931
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