Chlamydia pneumoniae Infection Increases Adherence of Mouse Macrophages to Mouse Endothelial Cells In Vitro and to Aortas Ex Vivo

Cells and animals.The buffers used were Hanks’ balanced salt solution (HBSS; NaCl, 8 g; glucose, 1 g; KCl, 0.4 g; KH₂PO₄, 60 mg; Na₂HPO₄, 48 mg; MgSO₄ · 7H₂O, 0.2 g; CaCl₂, 0.11 g [per liter, pH 7.2]), a glucose-potassium-sodium-phosphate (GKNP) solution (HBSS containing no Ca²⁺ or Mg²⁺), and chlamydia transport medium SPG (0.2 M sucrose, 0.8 mM KH₂PO₄, 6.7 mM Na₂HPO₄, 5 mM l-glutamic acid; pH 7.4). The culture media used were (i) RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum and 100 μg/ml each streptomycin and vancomycin and (ii) Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 5% fetal calf serum and 100 μg/ml each streptomycin and vancomycin. Cell lines used were a continuous human epithelial cell line (HL cells) obtained from Linda Cles (Department of Medicine, University of Washington, Seattle) and mouse endothelial cell line (MS1) from the American Type Culture Collection (Manassas, VA). MS1 is a pancreatic islet endothelial cell line transformed by retrovirus-mediated gene transfer in 1994 (1). The diets used were (i) regular chow and (ii) an atherogenic diet consisting of 15% fat, 1.25% cholesterol, and 0.5% sodium cholate (TD 88051; Harlan Teklad, Madison, WI). The mouse strains used were GFP transgenic mice (24) and ApoE (25) and ICAM-1 (13) knockout mice on a C57BL/6J background from Jackson Laboratories (Bar Harbor, ME).

Preparation of C. pneumoniae. C. pneumoniae strain AR-39 was propagated in HL cells and purified by Hypaque-76 (Nycomed Inc., Princeton, NJ) linear gradient centrifugation (16). Organisms purified by this method contain less than 0.1% host cell materials (15). Purified organisms (>1 × 10⁸ inclusion-forming units/ml) were resuspended in SPG buffer, aliquoted, and stored at −70°C until use. The infectivity titers were determined by titration in HL cells. Inoculum doses per macrophage or endothelial cell were expressed as multiplicities of infection (MOIs).

Preparation of mouse peritoneal macrophages.GFP transgenic mice were injected intraperitoneally with 1 ml of 3% thioglycolate medium. After 4 to 5 days, peritoneal macrophages were harvested by injection of 10 ml of warm GKNP. The abdomen was massaged, and peritoneal fluids were withdrawn into a 50-ml tube and kept on ice. The macrophage suspension was centrifuged (700 × g, 5 min, 4°C) and resuspended in RPMI 1640 medium supplemented with 10% fetal calf serum. Peritoneal macrophages were plated into six-well plates. After adsorption at 37°C for 2 h in 5% CO₂, the plates were washed once with RPMI 1640 medium to remove unattached cells. Adherent macrophages were cultured for 1 or 2 days at 37°C in 5% CO₂ before use.

Infection of macrophages.Murine peritoneal macrophages cultured for 1 day were used. Macrophages were inoculated with C. pneumoniae in 2 ml of RPMI 1640 medium by centrifugation at 700 to 800 × g at room temperature for 1 h. Inoculated cells were incubated for 1 h at 37°C in 5% CO₂. Cells were washed once with 2 ml of RPMI 1640 medium and incubated with RPMI 1640 medium supplemented with 10% fetal calf serum for 1 day.

Infection of MS1 cells.MS1 cells were infected with C. pneumoniae by the same method described above for macrophages, with minor modifications. MS1 monolayers on 24-well plates were inoculated with various doses of C. pneumoniae in 0.5 ml of DMEM, centrifuged at 700 to 800 × g at room temperature for 1 h, and incubated for 1 h at 37°C in 5% CO₂. Cells were washed once with 1 ml of DMEM and incubated with DMEM supplemented with 5% fetal calf serum for 1 day.

In vitro adhesion assay.MS1 cells were seeded onto a 24-well plate 1 to 2 days before inoculation. Murine macrophages cultured in six-well plates were washed twice with GKNP and harvested by scraping with a rubber policeman. Harvested macrophages were centrifuged at 700 × g for 5 min at 4°C. Macrophage pellets were resuspended with HBSS. Each MS1 monolayer was inoculated with 0.5 ml of a suspension of 3 × 10⁴ macrophages at 37°C with rocking. After 30 min of adsorption, the plate was washed twice with HBSS to remove nonadherent macrophages and fixed with 10% buffered formalin for 30 min at 4°C. The adherent macrophages were quantified by a fluorescence microscope at a magnification of ×200 with a fluorescein isothiocyanate-based filter. Twenty fields were counted per well. The results were expressed as the mean ± standard deviation of three or four wells.

Ex vivo adhesion assay.Ex vivo adhesion assays were performed as described by Ishida et al. (9). Briefly, the thoracic aortas of 13-week-old female mice were isolated and pinned on a 3% agarose-coated six-well plate with fine needles. Macrophages (2 × 10⁵ to 5 × 10⁵) left uninfected or infected with C. pneumoniae in HBSS at an MOI of 0.5 were inoculated onto each aortic strip. The plate was rocked gently for 30 min at 37°C. Unbound macrophages were removed by washing twice with HBSS. Bound macrophages were fixed with 10% buffered formalin for 30 min at 4°C. The bound macrophages were counted.

Assay of plasma cholesterol.Blood was collected in heparinized tubes. Plasma was separated from blood cells by centrifugation and frozen at −70°C until tested. Cholesterol levels were measured by a commercial enzymatic test kit (Sigma, St. Louis, MO). Cholesterol levels were determined in triplicate and averaged.

Statistical analysis.Data are expressed as the mean ± the standard deviation. An unpaired two-tailed Student t test was used for determination of statistical significance. The P value was determined with Microsoft Excel (Microsoft, Redmond, WA).

Adherence of C. pneumoniae-infected macrophages to endothelial cells in vitro. C. pneumoniae infection of macrophages was shown to stimulate adherence of macrophages to uninfected endothelial cells. Figure 1A shows adherent green macrophages under a fluorescence microscope. The increase in the number of adherent green macrophages in the infected group (infected at an MOI of 0.1) over the uninfected group can be readily discerned (Fig. 1A). In addition, in the infected group, clusters of two to four macrophages were observed. Quantification of adherent macrophages showed significant increases of 2.0- to 3.8-fold in the number of macrophages that adhered to endothelial cells when macrophages were infected at MOIs of 0.1, 0.5, 1.0, and 10 (Fig. 1B; done in triplicate).

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FIG. 1.

Adherence of C. pneumoniae-infected mouse peritoneal GFP-macrophages to murine endothelial cells (MS1). (A) Fluoroscopic image showing that infected GFP-macrophages adhere better than uninfected macrophages to MS1. (B) Determination of the infectious dose (MOI) required for promotion of adherence. Macrophages were infected with C. pneumoniae at an MOI of 0.1 (A) or at various MOIs (B) for 1 day and assayed for adherence. Macrophages (3 × 10⁴ per well) were added to MS1 monolayers, incubated for 30 min, and washed, and adherent macrophages were counted with a fluorescence microscope at a magnification of ×200 with a fluorescein isothiocyanate-based filter. *, P < 0.01 (MOIs of 0.5, 1.0, and 10) and P < 0.02 (MOI of 0.1).

Adherence of macrophages to C. pneumoniae-infected endothelial cells in vitro. C. pneumoniae infection of endothelial cells was shown to increase the adherence of uninfected macrophages from female mice to endothelial cells. Endothelial cells were infected at MOIs of 0.1, 0.5, 1, and 10 (Table 1). Significant adherence increases of 2.0- and 1.8-fold were observed at MOIs of 1 and 10, respectively (P < 0.01). A similar magnitude of enhancement was observed when GFP-macrophages from male mice were used. The increases were 1.7- and 2.3-fold at MOIs of 1 and 10, respectively (P < 0.01 and P < 0.05).

Adherence of C. pneumoniae-infected macrophages to the aortas of normo- and hyperlipidemic mice ex vivo. C. pneumoniae infection of macrophages resulted in significant increases in the adherence of macrophages to the aortas of normolipidemic C57BL/6J mice fed a chow diet (1.4-fold, Fig. 2A), hyperlipidemic ApoE-deficient mice fed a chow diet (1.3-fold, Fig. 2A), and C57BL/6J mice fed an atherogenic diet (1.6-fold, Fig. 2B). The adherence of uninfected macrophages to the aortas of hyperlipidemic, ApoE-deficient mice in comparison to those of normolipidemic C57BL/6J mice was also shown to increase slightly, but the increase was not statistically significant (Fig. 2A). The plasma cholesterol levels were 54 ± 7 mg/dl in C57BL/6J mice fed a chow diet, 310 ± 41 mg/dl in ApoE-deficient mice fed a chow diet, and 112 ± 22 mg/dl in C57BL/6J mice fed an atherogenic diet.

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FIG. 2.

Adherence of C. pneumoniae-infected mouse peritoneal GFP-macrophages to the murine aorta. Mouse aortic strips were isolated from 13-week-old female C57BL/6J and ApoE^−/− mice fed a chow diet (A) and C57BL/6J and ICAM-1 knockout mice fed an atherogenic diet for 5 weeks (B). The aortas were incubated with murine peritoneal GFP-macrophages. The adherent macrophages were counted under a fluorescence microscope. Data are presented as the percentage of adherent macrophages compared to that of the control (uninfected C57BL/6J mice). *, P < 0.05.

Adherence of C. pneumoniae-infected macrophages to the aortas of ICAM-1 knockout mice ex vivo.To study the role of adhesion molecules in C. pneumoniae-associated atherosclerosis, the adherence of C. pneumoniae-infected macrophages to the aortas of ICAM-1 knockout mice fed an atherogenic diet was evaluated. The plasma cholesterol levels of ICAM-1 knockout mice were 159 ± 12 mg/dl. Infected macrophages adhered no better than uninfected macrophages to the aortas of ICAM-1 knockout mice (Fig. 2B). This was in contrast to the 1.6-fold increase in adherence (P < 0.05) observed with C. pneumoniae-infected macrophages in comparison to uninfected macrophages in hyperlipidemic C57BL/6J mice (Fig. 2B). These findings indicate that ICAM-1 may play a role in C. pneumoniae-accelerated atherosclerosis. In contrast, no decrease in the adherence of uninfected macrophages was observed in ICAM-1-deficient mice in comparison to C57BL/6J mice. This finding may suggest a compensatory effect from other adhesion molecules which were activated in ICAM-1 knockout mice by hyperlipidemia induced by the atherogenic diet (4). Hyperlipidemia has been shown to activate the expression of endothelial adhesion molecules VCAM-1 and ICAM-1 (8).