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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2003;23:789.)
© 2003 American Heart Association, Inc.

Vascular Biology

Recruitment of Chlamydia pneumoniae–Infected Macrophages to the Carotid Artery Wall in Noninfected, Nonatherosclerotic Mice

Andreas E. May; Vanessa Redecke; Sabine Grüner; Roland Schmidt; Steffen Massberg; Thomas Miethke; Birgit Ryba; Clarissa Prazeres da Costa; Albert Schömig; Franz-Josef Neumann

From Deutsches Herzzentrum und 1. Medizinische Klinik (A.E.M., V.R., S.G., R.S., S.M., B.R., A.S.) and Institut für Mikrobiologie, Immunologie und Hygiene (V.R., T.M., C.P.d.C.), Klinikum Rechts der Isar, Technische Universität München and Herz-Zentrum Bad Krozingen (F.-J.N.), Germany.

Correspondence to Andreas E. May, MD, Deutsches Herzzentrum, Technische Universität München, Lazarettstr. 36, D-80636 München, Germany. E-mail may{at}dhm.mhn.de

	Abstract

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Objective— Monocyte recruitment into the subendothelium is a crucial step in atherogenesis. Chlamydia pneumoniae resides in circulating monocytes and in the atherosclerotic vascular wall. However, the role of C pneumoniae for monocyte recruitment is unknown. The aim of this study was to examine the impact of C pneumoniae on monocyte adhesion and migration.

Methods and Results— C pneumoniae–infected, fluorescence-labeled mouse macrophages (ANA-1) were injected intravenously into noninfected, healthy mice. In vivo videomicroscopy showed increased rolling and firm adhesion to the carotid artery compared with noninfected macrophages. In vitro, C pneumoniae infection (yielding 25% to 35% infected monocytes) increased adhesion of human monocytes or MonoMac6 cells to human umbilical vein endothelial cells and improved cell migration through endothelial-like ECV604 cells. Cell adhesion was inhibited by antibody blockade of very late antigen-4, lymphocyte function-associated antigen-1, macrophage antigen-1, or urokinase receptor, which were found upregulated or activated on C pneumoniae infection (flow cytometry). In contrast, C trachomatis did not induce monocyte adhesion at comparable infection rates (25% to 35%), indicating a unique activation pathway for C pneumoniae. Polymyxin B did not affect C pneumoniae–induced adhesion, excluding a relevant role of lipopolysaccharide in this process.

Conclusions— These data indicate that C pneumoniae can direct monocytes to predilection sites of nonatherosclerotic vessel walls in vivo by activation of the integrin adhesion receptor system.

Key Words: Chlamydia pneumoniae • monocyte • adhesion • integrin • atherosclerosis

	Introduction

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Atherosclerosis is an inflammatory disease that is initiated and accelerated by early infiltration of monocytes.^1,2 Epidemiological and animal studies suggest a pathophysiological role for Chlamydia pneumoniae in the development and progression of atherosclerosis.^3–6 Being an obligate intracellular bacterium, C pneumoniae has been detected in circulating monocytes and atherosclerotic lesions^7,8 and can activate inflammatory processes in endothelial and smooth muscle cells in vitro.^9–11 However, it is unknown how C pneumoniae is directed into the subendothelium to execute its inflammatory potential.⁶ Recent studies suggest that monocytes may act as vectors and systemically disseminate C pneumoniae.^12,13 However, to adhere and migrate through the vessel wall, monocytes have to go through a highly coordinated process, which requires the activation of different adhesion receptors in a cascade-like fashion.¹⁴

The integrin family of adhesion receptors is critically involved in this process by mediating cell-to-cell and cell-to-extracellular matrix contacts.¹⁴ On cell activation, integrins become quantitatively upregulated and undergo conformational changes, resulting in a ligand binding state. The integrin very late antigen-4 (VLA-4, ₄ß₁) mediates monocyte tethering and firm adhesion to endothelial vascular cell adhesion molecule-1 but additionally initiates tight cell adhesion via downstream activation of the two ß₂-integrins lymphocyte function-associated antigen-1 (LFA-1, _Lß₂) and macrophage antigen-1 (Mac-1, _Mß₂).¹⁵ Integrins form complexes with the urokinase receptor (uPAR),^16,17 which is required for ß₂-integrin–mediated leukocyte emigration.¹⁸

In this study, we provide evidence that infection with C pneumoniae can initiate rolling and adhesion of macrophages to the noninflamed vessel wall at predilection sites of noninfected, nonatherosclerotic mice. We demonstrate in vitro that C pneumoniae–infected monocytic cells show enhanced transmigration and attach to the endothelium via the activated integrins VLA-4, LFA-1, and Mac-1, involving uPAR. These experiments indicate that and by what means C pneumoniae–infected monocytes may be armed to invade the not (yet) inflamed subendothelium and initiate inflammatory processes. Moreover, our data indicate a unique activation pathway for C pneumoniae, which might be operative in the aggravation of atherosclerosis.

	Methods

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Reagents
Phorbol 12-myristate 13-acetate (PMA), Escherichia coli lipopolysaccharide, and polymyxin B were from Gibco. Fibrinogen and fibronectin were from Sigma. Vitronectin was provided by Dr K.T. Preissner (Giessen, Germany). Recombinant monocyte chemoattractant protein-1 was from R&D. FITC-conjugated mAb anti-Chlamydia pneumoniae (ACI) was from Progen. mAbs mouse anti-human CD29 (ß₁-integrin chain, K20), CD49d (₄-chain, blocking, HP2.1), CD11b (_M-chain, Bear1), and CD62P (P-selectin, clone CLBThrombo/6) were from Immunotech; mAb mouse anti-human CD18 (ß₂-chain, blocking, IB4) was from Ancell. Mab mouse anti-human CD11a (_L-chain, blocking, L15) was kindly provided by Dr C. Figdor (Nijmegen, The Netherlands). mAb CBRM1/5, which recognizes only the activated epitope of CD11b and blocks Mac-1–dependent adhesion,¹⁹ was generously provided by Dr T. Springer (Boston, Mass). mAb24 detects another activation-dependent epitope on LFA-1 and Mac-1 and was a kind gift from Dr N. Hogg (London, England).¹⁴ mAbs anti-uPAR R3 (blocking) and R4 (nonblocking) were kindly provided by Dr G. Hoyer-Hansen (Copenhagen, Denmark). Murine anti-human IgG2a (Sigma) was used as an isotype-matched control antibody.

Cells
Human MonoMac6 cells, which represent monocytic cells with a closely related pattern of surface receptors and monocyte-like behavior,²⁰ were cultured in VLE-RPMI-1640 (Biochrom) containing 10% low-tox FCS (Clonetics).

For control experiments, freshly isolated human monocytes were used. Mononuclear cells were isolated by centrifugation of whole CPDA-anticoagulated blood on a Ficoll gradient (20 minutes, 800g, 4°C). Mononuclear cells were cultured on plastic dishes for 12 hours (0.5x10⁶/mL) in RPMI containing 10% FCS, and nonadherent cells were removed by gentle washing. The remaining cells (85% to 90% monocytes) were resuspended using EDTA (0.05% PBS) and washed in RPMI before chlamydial infection.

Human umbilical vein endothelial cells (HUVECs) were purchased (Clonetics) and cultured (for 2 to 4 passages) in low serum endothelial cell growth medium (PromoCell) on gelatin-coated tissue-culture plastic.

ECV604 (ATCC) expresses an endothelial-like pattern of adhesion receptors and was cultured in RPMI-1640 containing 10% FCS (Gibco). The murine macrophage cell line Ana-1²¹ (ATCC) was cultivated in DMEM containing 10% FCS.

Infection Protocol
C pneumoniae CM1 (ATCC; VR-1360) and C trachomatis (L2, ATCC, VR-902B) were cultured and purified as described.^11,13 Briefly, chlamydial elementary bodies were propagated in HEp-2 monolayers for 72 hours in the presence of cycloheximide (1 µg/mL, Sigma). The harvested cells were disrupted with glass beads and the elementary bodies purified by sucrose-urografin gradient and stored in liquid nitrogen. The number of inclusion-forming units in HEp-2 cells was quantified by staining with the chlamydia-specific antibody followed by fluorescence microscopy. In most experiments, monocytic cells were washed twice, resuspended at a density of 250 000 cells/mL, and inoculated with 5 inclusion-forming units of C pneumoniae per cell for 48 hours, leading to 25% to 35% (multiplicity of infection, 0.25 to 0.35) of cells. Successful infection was routinely confirmed by staining for C pneumoniae with the fluorescence-conjugated antibody followed by fluorescence microscopy and flow cytometry. In some experiments (see Figure 2C), isolated human monocytes were infected with increasing concentrations of C pneumoniae or C trachomatis followed by quantification of infected monocytes as well as of cell adhesion to endothelial cells.

View larger version (45K): [in this window] [in a new window]   Figure 2. C pneumoniae induces monocyte adhesion and transmigration in vitro. MonoMac6 cells were pretreated with medium, infected with C pneumoniae for 48 hours as described in the Methods section, or stimulated with PMA (10 ng/mL, 60 minutes); after washing twice, cell adhesion to HUVEC monolayers (A) and transmigration through monolayers of endothelial-like ECV604 cells (B) were performed as described in the Methods section. C, Isolated human monocytes were pretreated with medium or infected with increasing concentrations of C pneumoniae or C trachomatis for 48 hours, as described in the Methods section, leading to different monocyte infection rates as indicated, which were quantified by fluorescence microscopy using fluorescence-labeled mAb anti-chlamydia-LPS. After washing twice, cell adhesion to HUVEC monolayers was performed. Values represent the mean±SD of 18 (A), 5 (B), and 4 (C) independent experiments. *P<0.05 compared with the medium-treated control.

Adhesion and Transmigration Assays
Adhesion and transmigration assays were performed as described.^15,22,23

Cell-to-Cell Adhesion
HUVECs were seeded onto gelatin-coated 96-well plates 48 hours before the experiment. Confluency was confirmed by microscopic inspection before each experiment. Monocytic cells were pretreated (medium, C pneumoniae infection, C trachomatis infection, or PMA stimulation) as described in the figure legends, respectively. The cells were washed twice in adhesion medium (serum-free RPMI 1640/HEPES 25 mmol/L) and coincubated (7x10⁵/mL) with HUVEC monolayers in the absence or presence of blocking mAbs (20 µg/mL) directed against various adhesion receptors (see figure legends). After 30 minutes of coincubation (37°C, 5% CO₂, 90% humidity), the plates were gently washed twice to remove nonadherent cells, and adherent monocytes were quantified by counting 16 high-power fields using light microscopy. At least triplicate wells were run per test substance. The mean of adherent cells that were treated by the respective control substance (medium) was equated with 100% of cell adhesion. Cell adhesion that was achieved by test substances was compared with this value.

Cell Adhesion to Immobilized Integrin Ligands
Ninety-six-well plates were coated with human fibrinogen, fibronectin, or vitronectin (each 20 µg/mL) for 2 hours at 37°C and blocked with 1% BSA (30 minutes, 25°C). Noninfected or C pneumoniae–infected leukocytes (see above) were seeded at 70 000 cells/well for 30 minutes (37°C, 5% CO₂, 90% humidity). After removal of nonadherent cells by two washing steps, cell adhesion was quantified by peroxidase reaction using p-nitrophenol as a substrate in an ELISA reader (BioRad).

Transmigration of Monocytic Cells
Before the experiment, ECV604 cells were seeded onto polycarbonate membranes (5.0-µm pore size) of transwell inserts that closely fitted into 48-well plates (Nunc) and were grown until confluency. MonoMac6 cells (2.5x10⁵/mL) that had been pretreated with medium orC pneumoniae (5 inclusion-forming units/cell) for 48 hours or with PMA during the last 60 minutes were washed twice, resupended (0.5x10⁶/mL), and placed into the upper compartment of the transwells. In parallel, medium containing monocyte chemoattractant protein-1 (10 µg/mL) was introduced into the lower compartment beneath the filter. After 12 hours, migrated monocytic cells were harvested from the lower compartment and quantified using a cell counter.

Flow Cytometry
Flow cytometry was performed as described.¹⁵ Briefly, cells were washed twice, resuspended (10⁶/mL), incubated with saturating concentrations of primary mouse anti-human mAbs (30 minutes, reverse transcriptase), washed again, and incubated with a secondary phycoerythrin-conjugated anti-mouse mAb (Immunotech, 30 minutes, room temperature). After washing, cells were analyzed on a FACScalibur flow cytometer (Becton Dickinson). Nonspecific fluorescence was determined by using isotype-matched mouse IgG as control primary mAb.

Reverse Transcriptase–Polymerase Chain Reaction
Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen). Contaminating DNA was removed using Message Clean kit (Gene Hunter). RNA was transcribed to cDNA using Omniscript reverse transcriptase (Qiagen) and random hexamers (Gibco). Polymerase chain reaction was performed with the HotStarTaqTM DNA Polymerase (Qiagen). Annealing temperature was 63°C. Primer sequences were as follows: uPAR (forward) 5'-GCCCTGGGACAGGACCTCTG-3', uPAR (reverse) 5'-CATTGATTCATGGGGCCTCGGC-3', Cyclophillin (forward) 5'-CATCTGCACTG-CCAAGACTG-3', Cyclophillin (reverse) 5'-CTGCAATCCAGCTAGGCATG-3'.

Intravital Microscopy
Ten-week-old male C57BL/6J mice (Charles River) were anesthetized, and the common carotid artery was dissected free from surrounding tissue. Murine Ana-1 macrophages were pretreated (medium, C pneumoniae infection, or PMA stimulation) as described in the figure legends, washed, and incubated with 0.02% rhodamine 6G (Molecular Probes). After washing twice, 1x10⁶ fluorescent cells were infused into the right jugular vein. Labeled cells were visualized in situ by intravital videomicroscopy of the right common carotid artery using a Zeiss Axiotech microscope (20x water immersion objective, W 20x/0.5) with a 100W HBO mercury lamp for epi-illumination. All videotaped images were evaluated using a computer-assisted image analysis program (Cap Image 7.1, Dr Zeintl, Heidelberg, Germany). Rolling macrophages were defined as cells crossing an imaginary perpendicular through the vessel at a velocity significantly lower than the centerline velocity; numbers represent cells per square millimeter of endothelial surface. Adherent cells were quantified by counting the cells that did not move or detach from the endothelial surface within 20 seconds.

Statistical Analysis
Comparisons between group means were performed using ANOVA. Data represent mean±SD. A value of P<0.05 was regarded as significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
C pneumoniae Induces Macrophage Rolling and Adhesion In Vivo
We hypothesized that C pneumoniae infection can stimulate monocyte recruitment to the nonatherosclerotic vessel wall under physiological flow conditions with high shear stress. The mouse macrophage cell line ANA-1 was infected with C pneumoniae for 48 hours, as described in the Methods section, washed, fluorescently stained with rhodamine, and injected into the right jugular vein of anesthetized noninfected, healthy mice. Fluorescent macrophages were visualized in situ in the right common carotid artery by intravital videomicroscopy. This setting allows the discrimination between cell rolling along the vessel wall and subsequent firm adhesion.

Only a few (if any) noninfected macrophages attached to the endothelium. In contrast,C pneumoniae–infected macrophages showed both enhanced rolling as well as firm adhesion to the vessel wall (Figures 1A and 1B), which were comparable to the maximum level achieved with PMA stimulation. Figure 1C shows representative photomicrographs of nonadherent, noninfected macrophages (left panel) and adherent, C pneumoniae–infected macrophages (right panel).

View larger version (62K): [in this window] [in a new window]   Figure 1. C. pneumoniae induces macrophage recruitment to the carotid artery in vivo. Murine Ana-1 macrophages were pretreated with medium, infected with C pneumoniae for 48 hours as described in the Methods section, or stimulated with PMA (10 ng/mL, 60 minutes). After washing twice, the cells were stained with 0.02% rhodamine (30 minutes, 37°C) and washed twice, and 1x106 fluorescent macrophages were infused into the right jugular vein of healthy C57BL/6J mice. Rolling (A) and firm adhesion (B) of the infused fluorescent cells were visualized in situ by in vivo videomicroscopy of the right common carotid artery and quantified using a computer-assisted analysis program. Values represent mean±SD of 3 independent experiments. *P<0.05. C, Representative photomicrographs of the vessel wall showing low adhesion of medium-treated, noninfected macrophages (left) and adherent, C pneumoniae–infected macrophages (right).

C pneumoniae Induces Monocytic Cell Adhesion and Transmigration In Vitro
The underlying mechanisms of chlamydia-mediated cell adhesion were analyzed in vitro. Human monocytic MonoMac6 cells were infected with C pneumoniae for 48 hours and washed, and cell adhesion to human umbilical vein endothelial cell monolayers (HUVEC) was studied. Adhesion of C pneumoniae–infected cells was significantly enhanced to an extent that was comparable to maximum cell stimulation with PMA (Figure 2A). Consistently, transmigration through confluent monolayers of endothelial-like ECV604 cells was significantly induced by C pneumoniae (Figure 2B). In addition, we have compared the effects of C pneumoniae to C trachomatis. Human isolated monocytes were infected with increasing concentrations ofC pneumoniaeorC. trachomatis, leading to increasing rates of cell infection. Figure 2C demonstrates that C trachomatis did not induce monocyte adhesion at infection rates of 30% of cells (multiplicity of infection, 0.3), a rate that allowed strong proadhesive effects of C pneumoniae. C trachomatis only induced monocyte adhesion when monocytes were infected with high concentrations of C trachomatis, leading to >70% of infected monocytes (multiplicity of infection, 0.7 to 0.9). Therefore, C pneumoniae seems to be specifically equipped to stimulate monocyte adhesion.

C pneumoniae Induces Monocytic Cell Adhesion via VLA-4, LFA-1, Mac-1, and uPAR
To identify the adhesion receptors involved, MonoMac6 cells were infected withC pneumoniaefor 48 hours and washed, and receptor-specific cell adhesion to immobilized extracellular matrix proteins was studied. C pneumoniae significantly induced monocytic cell adhesion to fibrinogen (ligand of Mac-1), fibronectin (ligand of VLA-4), as well as adhesion to vitronectin (ligand of uPAR) (Figure 3A). In addition, adhesion of C pneumoniae–infected monocytic cells to endothelial monolayers was performed in the presence of blocking antibodies directed against various adhesion receptors (Figure 3B). C pneumoniae–induced adhesion was effectively reduced by blockade of the ₄-integrin chain of VLA-4 (53% inhibition), which is known to mediate both cell rolling and firm adhesion. In addition, cell adhesion was reduced by inhibition of the respective -chains of LFA-1 (_L, 33% inhibition), of Mac-1 (_M, 56% inhibition), or of the common ß₂-integrin chain (65% inhibition). Furthermore, antibody blockade of uPAR, which is an essential coreceptor for ß₂-integrins, also inhibited cell adhesion to HUVECs. Notably, the extent of inhibition by uPAR blockade was comparable to the extent (54% inhibition) achieved by ß₂-integrin blockade. A nonspecific control mAb (anti–P-selectin) had no inhibitory effect. Together, several adhesion receptors that are well-known to act in concert in cell rolling or firm adhesion seem to cooperate in mediating adhesion of C pneumoniae–infected monocytes.

View larger version (20K): [in this window] [in a new window]   Figure 3. C pneumoniae induces monocytic cell adhesion via VLA-4, LFA-1, Mac-1, and uPAR. MonoMac6 cells were pretreated with medium or infected with C pneumoniae for 48 hours, as described in the Methods section, and washed twice. A, Cell adhesion to wells that were precoated with fibronectin (fn), fibrinogen (fg), or vitronectin (vn) (20 µg/mL each), as described in the Methods section. Values represent mean±SD of the increase in cell adhesion compared with noninfected cells (dotted line) and represent 10 independent experiments. *P<0.05 compared with control (noninfected, dotted line). B, Cell adhesion of C pneumoniae–infected MonoMac6 cells to HUVECs in the presence or absence of blocking mAbs anti-ß2-integrin (clone IB4), anti-L-integrin (clone L15), mAb anti-M-integrin (clone CBRM1/5), anti-4-integrin(clone HP2.1), and anti-uPAR (clone R3) or anti-P-selectin (clone CLBThrombo/6) (each 20 µg/mL). Values represent mean±SD of 5 independent experiments. *P<0.05 compared with adhesion of C pneumoniae–infected cells in the absence of any antibodies.

C pneumoniae Upregulates and Activates VLA-4 and ß₂-Integrins
Consistent with blocking experiments, flow cytometric analysis of C pneumoniae–infected monocytic cells revealed quantitative upregulation of VLA-4 and Mac-1 by using antibodies that detect the respective integrins independent from their functional state (Figure 4A). The expression of both the ₄-integrin (mean fluorescence 19±7 versus 10±4, n=10, P<0.01) and ß₁-integrin chain (mean fluorescence 115±68 versus 65±39, n=9, P<0.01) of VLA-4 was significantly upregulated on C pneumoniae–infected cells compared with noninfected cells. Similarly, both the _M-integrin (mean fluorescence 18±10 versus 9±3, n=9, P<0.01) and ß₂-integrin chain (mean fluorescence 19±11 versus 11±4, n=9, P<0.01) of Mac-1 were increased on C pneumoniae–infected cells.

View larger version (31K): [in this window] [in a new window]   Figure 4. C pneumoniae upregulates or activates VLA-4, LFA-1, and Mac-1. MonoMac6 cells were pretreated with medium (thin line) or infected with C pneumoniae (bold line) for 48 hours, as described in the Methods section, washed, and analyzed by flow cytometry. A, Quantitative integrin expression using mAbs anti-ß1-integrin (clone K20), anti-4-integrin (clone HP2.1), anti-ß2-integrin (clone IB4), and anti-M-integrin (clone Bear1), which detect the respective receptor independent of its functional state. B, Qualitative receptor activation using mAbs anti-4-integrin(clone HUTS), anti-M-integrin (clone CBRM1/5), and anti-L/M-integrin (clone mAb24) that only detect the respective receptor in its activated, ligand binding state. The dotted line represents the respective nonspecific control mAb. One representative histogram of 7 independent experiments is shown, respectively.

In addition, a functionally active state of VLA-4, Mac-1, and LFA-1 was noted on C pneumoniae–infected cells by using the respective mAbs HUTS (mean fluorescence 12±1 versus 7±1, n=7, P<0.02), CBRM1/5 (mean fluorescence 13±5 versus 7±2, n=11, P<0.01), and mAb24 (mean fluorescence 12±4 versus 7±2, n=10, P<0.01) that exclusively bind to the respective integrin in its activated, ligand-binding state (Figure 4B).

C pneumoniae Upregulates uPAR Expression
uPAR is known to become upregulated on monocytes in vitro on inflammatory stimuli, such as tumor necrosis factor- or interleukin-1ß,²⁴ or in vivo in acute myocardial infarction.²³ Previously, ß₂-integrin–mediated monocyte adhesion to the endothelium has been shown to depend on the presence of uPAR.^16,18,23 Thus, upregulation of uPAR may allow the formation of additional functional integrin/uPAR units, resulting in enhanced integrin activation. In accordance with enhanced cell adhesion to the uPAR-ligand vitronectin (Figure 3A), monocytic cell infection with C pneumoniae resulted in upregulation of uPAR on both surface protein (mean fluorescence 29±25 versus 18±19, n=12, P<0.01) and mRNA level (Figure 5).

View larger version (24K): [in this window] [in a new window]   Figure 5. C pneumoniae upregulates uPAR expression. MonoMac6 cells were pretreated with medium or infected with C pneumoniae for 48 hours, as described in the Methods section. A, Flow cytometric analysis of uPAR surface expression on C pneumoniae–infected (bold line) and noninfected (thin line) cells using mAb anti-uPAR (clone R4). The dotted line represents the nonspecific control mAb. One representative histogram of 12 independent experiments is shown. B, Analysis of uPAR mRNA expression in noninfected and C pneumoniae–infected cells by reverse transcriptase–polymerase chain reaction. This experiment has been repeated twice with comparable results.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Monocytes, adhering and infiltrating the vessel wall, play a pivotal role in the initiation and acceleration of atherosclerosis.^1,2 C pneumoniae is preferably found in atherosclerotic lesions, where it is suggested to induce atherogenic reactions in cells of the vascular wall, including macrophages, endothelial cells, and smooth muscle cells. This study describes a potential mechanism of how C pneumoniae may accelerate atherosclerosis in early stages by initiation of monocyte recruitment. C pneumoniae–infected mouse macrophages, which were intravenously injected into noninfected, healthy mice, showed enhanced rolling and firm adherence to the vascular wall of the carotid artery bifurcation. Being continuously exposed to high shear stress, the carotid artery represents a predilection site for atherosclerosis that has been shown to harbor C pneumoniae within the lesions.^7,8 Thus, our data indicate that C pneumoniae can actively direct macrophages into the not (yet) inflamed subendothelium even in the absence of additional atherogenic factors. Therefore, monocytes and C pneumoniae appear to direct each other into the subendothelium and, thereby, may mutually support their inflammatory activities within the vessel wall.

Recently, enhanced adhesiveness of C pneumoniae–infected monocytes was shown using static adhesion assays in vitro.^25,26 Our study extends this concept by adding functional relevance in vivo and by providing the adhesive mechanism. Corresponding to the two C pneumoniae–activated processes in vivo, cell rolling and firm adhesion, the related receptors VLA-4, LFA-1, Mac-1, and uPAR were found upregulated or activated on C pneumoniae–infected cells. Consistently, C pneumoniae–induced adhesion was significantly inhibited by antibody blockade of these receptors, indicating that VLA-4, LFA-1, Mac-1, and uPAR cooperate in mediating C pneumoniae–induced monocyte adhesion. VLA-4 mediates the first physical contact with endothelial cells, a process called rolling.²⁷ VLA-4 engagement activates LFA-1 and Mac-1,¹⁵ which mediate firm cell adhesion and transmigration. Adequate LFA-1 and Mac-1 function requires the presence of uPAR,^16,28 which was found to be upregulated on C pneumoniae infection (Figure 5). Antibody blockade of uPAR inhibited monocytic cell adhesion to endothelial cells (Figure 3B) as well as to the Mac-1 ligand fibrinogen (not shown) to a comparable extent as achieved by ß₂-integrin inhibition, confirming our observations^15,18,23 and the observations of others²⁸ that uPAR is essential for ß₂-integrin–mediated adhesion. The pathophysiological relevance of these described adhesion receptors is supported by multiple studies showing that the respective function or dysfunction of one receptor has a direct impact on cardiovascular pathologies.^29,30

Our experimental setting of injection of ex vivo–infected macrophages into healthy mice allowed us to study interactions of an infected monocyte population with noninfected, healthy vessel walls in vivo. Notably, flow cytometric analysis revealed that the entire population within the group of infected cells showed upregulation and activation of adhesion receptors (see Figures 4 and 5). However, only 25% to 35% of those cells positively stained for C pneumoniae and thus could be considered infected. Thus, activation of noninfected cells may have occurred by soluble factors, such as cytokines. These data suggest that C pneumoniae–infected circulating monocytes may have the capacity to induce an adhesive phenotype in adjacent, noninfected monocytes.

Experimental and animal studies suggest that C pneumoniae, but not C trachomatis, exerts atherogenic activities in host cells, thereby accelerating atherosclerosis.^5,11 Hu et al⁵ have intranasally infected hypercholesterolemic mice withC pneumoniaeorC trachomatis. Only mice infected withC pneumoniaedisplayed enhanced atherosclerotic lesions. However, both C pneumoniaeand C trachomatisantigens were detected in the atherosclerotic aorta of mice infected with the corresponding bacteria. However, our study shows thatC pneumoniae–infected monocytes are armed to strongly adhere to the vascular endothelium, whereas C. trachomatis–infected monocytes only adhere when heavily infected. Together, these two studies support the concept of a unique atherogenic pathway for C pneumoniae. Moreover, these two studies suggest that C pneumoniae may actively contribute to macrophage recruitment and acceleration of atherosclerosis, whereas C trachomatis may become passively transported into the atherosclerotic vessel wall of hypercholesterolemic mice without actively influencing cell recruitment and atherogenesis. Whether host conditions such as hypercholesterolemia may preferably trigger C trachomatis–infected monocytes compared with noninfected monocytes to invade the inflamed vessel wall needs to be clarified by future studies.

The unique C pneumoniae–specific activation pathway has not been clarified at present. In our setting, chlamydial LPS did not play a predominant role for monocytic cell adhesion, because polymyxin B treatment had no inhibitory effect (not shown). Chlamydial heat shock protein 60 (cHSP60) has previously been shown to stimulate atherogenic properties in macrophages. In our study, cHSP60 enhanced monocytic cell adhesion by 1.8-fold (not shown), which could not fully account for the observed proadhesive effects of the bacterium. Thus, cHSP60 may cooperate with other, not yet identified stimulating components of the bacteria in the induction of the adhesive phenotype of monocytes.

In conclusion, C pneumoniae has the potential to induce a functionally active, adhesive state in monocytic cells by activation of the integrin adhesion receptor system. Our data indicate that C pneumoniae is not just transported into the subendothelium by monocytes as an innocent bystander but can actively contribute to the monocyte recruitment to predilection sites of atherosclerosis. Thereby, C pneumoniae may initiate inflammatory processes in the vascular wall during early stages of atherosclerosis.

	Acknowledgments

 
This work was supported by grants to A.E. May from the Gesellschaft für Thrombose and Hämostaseforschung (GTH) and the Wilhelm Sander-Stiftung (2000.113.1).

Received May 13, 2002; accepted March 10, 2003.

	References

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