Published online before print April 24, 2003, doi: 10.1161/01.RES.0000074001.46892.1C
© 2003 American Heart Association, Inc.
Cellular Biology |
Chlamydophila pneumoniae Induces ICAM-1 Expression in Human Aortic Endothelial Cells via Protein Kinase C–Dependent Activation of Nuclear Factor-
B
From the Department of Microbiology and Immunology (S.A.V.), Graduate Program in Molecular and Cellular Biology and Pathobiology (G.K.), and Division of Endocrinology-Metabolism-Nutrition (M.F.L.-V.), Medical University of South Carolina, and Ralph H. Johnson VA Medical Center (M.F.L.-V.), Charleston, SC.
Correspondence to Maria F. Lopes-Virella, MD, PhD, Ralph H. Johnson VA Medical Center and Medical University of South Carolina, Strom Thurmond Research Building, 114 Doughty St, Charleston, SC 29425. E-mail virellam{at}musc.edu
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Abstract |
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Chlamydophila pneumoniae has an epidemiological link with atherosclerosis and acute cardiovascular events. One mechanism that may explain such a link is the increased expression of intracellular adhesion molecule-1 (ICAM-1) in C pneumoniae–infected endothelial cells. Upregulation of ICAM-1 by C pneumoniae is well recognized and has been extensively studied, but the signaling pathways involved are not yet defined. Because upregulation of ICAM-1 by cytokines and other stimuli has been shown to be mediated by either mitogen-activated protein kinase, protein kinase C (PKC), or nuclear factor-
B (NF-B) pathways, we examined whether these pathways were involved in the ICAM-1 upregulation induced by C pneumoniae. Our data show a time-dependent phosphorylation of p44/p42 and SAPK/JNK pathways in C pneumoniae–infected cells. However, inhibition of the classic mitogen-activated protein kinase pathway using the PD98059 and U0126 inhibitors and inhibition of SAPK/JNK pathway did not suppress C pneumoniae–induced ICAM-1 expression. C pneumoniae also activates the NF-B pathway at 30 minutes after infection. Treatment of human aortic endothelial cells (HAECs) with the NF-B inhibitors BAY117085 and caffeic acid phenethyl ester led to a concentration-dependent inhibition of C pneumoniae–induced ICAM-1 upregulation. Finally, C pneumoniae–infected HAECs show membrane translocation of total PKC 30 minutes after cell infection. Calphostin C, a general PKC inhibitor, blocked both C pneumoniae–induced ICAM-1 expression and C pneumoniae–induced NF-B translocation. In conclusion, we demonstrated that C pneumoniae–induced ICAM-1 expression in HAECs requires NF-B and PKC activation and that NF-B activation is PKC dependent.
Key Words: Chlamydophila pneumoniae • intercellular adhesion molecule-1 • nuclear factor-B • protein kinase C • mitogen-activated protein kinase pathway
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Introduction |
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Intercellular adhesion molecule-1 (ICAM-1), a member of the immunoglobulin supergene family, plays a role in immune and inflammatory responses1 because of its critical role in mediating monocyte adhesion to the endothelium2 as well as transmigration of leukocytes. It also serves as a coactivation signal in T cell activation3 and in cell-matrix adhesive interactions.4 ICAM-1 expression is upregulated by several inflammatory mediators, such as cytokines (interleukin-1ß [IL-1ß], tumor necrosis factor [TNF], and interferon-
),5 oxidant stress,6 shear stress, and low-density lipoproteins (native LDL and oxidize LDL).7,8 In addition, ICAM-1 serves as a receptor for rhinoviruses,9 and it is upregulated by a variety of microorganisms, including Chlamydophila pneumoniae.10 ICAM-1 is minimally expressed in normal endothelium; however, in the earlier stages of atherosclerosis, significant upregulation of ICAM-1 expression is usually observed.11 C pneumoniae is an important respiratory pathogen associated with pneumonia, pharyngitis, bronchitis, and sinusitis.11 In the last decade, an association of C pneumoniae with atherosclerosis and coronary artery disease has been described by numerous investigators.12–15 C pneumoniae is able to replicate in macrophages, endothelial cells, and smooth muscle cells,16–18 the 3 major cell types present in atherosclerotic lesions. Infection of human endothelial cells with C pneumoniae has been shown to upregulate the expression of adhesion molecules, cytokines, and growth factors.19–21 It has been also shown that C pneumoniae, by leading to increased expression of cell adhesion molecules,10,22 induces transendothelial migration of neutrophils and monocytes22 and promotes activation of proteins with procoagulant activity.23 Regardless of the extensive literature on C pneumoniae–induced upregulation of ICAM-1, very little is known about the signal transduction pathways involved in the process.
Therefore, we decided to investigate the signaling pathways involved in C pneumoniae–induced ICAM-1 upregulation in human aortic endothelial cells (HAECs) to identify possible therapeutic targets to prevent ICAM-1 upregulation. Because upregulation of ICAM-1 by cytokines and other stimuli has been shown to be mediated by mitogen-activated protein kinase (MAPK), protein kinase C (PKC), or nuclear factor-B (NF-B) pathways,4 we decided to investigate whether some or all of the above pathways were involved in the ICAM-1 upregulation induced by C pneumoniae.
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Materials and Methods |
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Cell Cultures
Hep-2 cells (ATCC CCL23) were maintained in minimal essential medium (Sigma-Aldrich Co) containing Earle’s salts (EMEM) following ATCC recommendations. Primary HAECs were cultured following manufacturer’s recommendations (Cascade Biologics, Inc) and used in passages 2 to 6.
Chlamydophila pneumoniae Propagation
C pneumoniae AR39 (ATCC 53592) was propagated in Hep-2 cell monolayers as described before.24 After 72 hours of incubation, C pneumoniae was harvested by mechanical disruption, followed by a low-speed (250g) centrifugation. Elementary bodies present in the supernatant were pelleted at 30 000g (JA-20 rotor, Beckman Instruments, Inc) for 30 minutes. The pellets were suspended in 0.01 mol/L sodium phosphate (pH 7.2) containing 0.25 mol/L sucrose and 5 mmol/L L-glutamic acid (SPG)25,26 and stored at -70°C. HAECs were infected as previously described.24 In brief, HAECs grown in 6-well plates (2.5x105 cells/well) were infected with 2 mL of medium containing 6 to 10x105 inclusion forming units (IFUs). HAECs grown in 100-mm dishes (1.5x106 cells) were infected with 8 mL of medium containing 
16x106 IFU.
In some experiments, before addition to the cells, C pneumoniae was heat treated for 30 minutes at 95°C or UV irradiated for 30 minutes at 15 cm from the UV source (1200x100 mJ/cm2, Hoefer UVC500).21 Because the C pneumoniae inoculum may contain remnants of HEp-2 cells, we used as controls mock-infected cells (HAECs treated with crude preparations of noninfected HEp-2 cells processed in the same manner as infected Hep-2 cells). HAEC monolayers grown in coverslips were used to monitor the course of infection by immunofluorescence staining using a genus-specific fluorescein isothiocyanate (FITC)-labeled monoclonal antibody (Pathfinder Chlamydia confirmation system; Bio-Rad). Titration of C pneumoniae in infected HAECs was assessed according to previously published methodology.17
ICAM-1 Expression by Immunoblotting
HAECs were lysed with Triton X buffer (10 mmol/L HEPES, 200 mmol/L NaCl, 2 mmol/L CaCl2, 2.5 mmol/L MgCl2, 1.5% vol/vol Triton X) containing 100 µg/mL phenylmethylsulfonyl fluoride and 10 µg/mL leupeptin (Sigma-Aldrich Co). Cell lysates were centrifuged, and the protein concentration in the supernatant was determined using a BCA Protein Assay Reagent Kit (Pierce). Western blots were performed as described27 using an anti-hICAM-1 monoclonal antibody diluted at 1:500 (Calbiochem-Novabiochem). A horseradish peroxidase–conjugated anti-mouse antibody (Calbiochem-Novabiochem) was used as secondary antibody. ICAM-1 was visualized after incubation of the membranes in Chemiluminescence Reagent Plus (Perkin Elmer Life Science, Inc) followed by exposure to X-ray film.
In some experiments, HAECs were incubated for 30 to 60 minutes with inhibitors of the several signaling pathways studied before C pneumoniae infection. As inhibitors for the MAPK pathways, the MEK1/2 inhibitor U0126 (Cell Signaling Technology), the MEK1 inhibitor PD98059, and the Jun N-terminal kinase (JNK) inhibitor SP600125 (Calbiochem-Novabiochem) were used. BAY11-7085 and caffeic acid phenethylester (CAPE) were used to inhibit the NF-B pathway, and calphostin C, bisindolylmaleimide I, and Gö 6976 were used to inhibit PKC pathway. All of the above inhibitors were from Calbiochem-Novabiochem.
The toxicity to HAECs of all the inhibitors used was determined by cell morphology and trypan blue dye exclusion of trypsinized cells using different concentrations of the drugs. Concentrations not toxic to HAECs were chosen.
Northern Blot Analysis
ICAM-1 cDNA (1 mg/mL) was generously provided by Dr Jeffrey Greve (Bayer Pharmaceutical Division, Berkeley, Calif). The cDNA probe was prepared by polymerase chain reaction (PCR) using a Qiagen Taq DNA Polymerase Kit (Qiagen). The following primers were used: 5'-GAGATCACCATGGAGCCAAT-3'/5'-GGG-CCTCACACTTCACTGTC-3' (Sigma-Genosys).
Total cellular RNA was isolated from HAECs using an RNeasy Mini kit (Qiagen) following the instructions from the manufacturer. Purification and quantification of RNA were assessed by A260/A280 absorption, and an aliquot of RNA (10 µg) from samples with an absorbance ratio above 1.6 was fractionated using a 1.2% agarose formaldehyde gel. Northern blotting of ICAM-1 mRNA was performed as previously described.28
C pneumoniae and Mitogen-Activated Protein Kinase Pathway
Cell protein lysates were prepared as described in previous sections. Immunoblotting was performed using PhosphoPlus P44/42, p38, and SAPK/JNK antibody kits (Cell Signaling Technology) and following the manufacturer’s instructions.
C pneumoniae and NF-B Pathway
Nuclear extracts were prepared from infected or controls confluent HAECs (1.5x106 cells) as described before.27,29 Forty micrograms of protein were electrophoresed under reducing conditions on a 10% SDS-polyacrylamide gel. Immunoblotting was performed using an antibody (1 µg/mL) to the activated p65 subunit of NF-B (Zymed). Immunoblotting of phosphorylated IB-
was performed using an antibody at 1:100 dilution against human IB- that contains the phosphorylated Ser-32 amino acid (Santa Cruz Biotechnology).
Electrophoretic Mobility Shift Assay
To determine nuclear translocation and binding of NF-B, electrophoretic mobility shift assay (EMSA) was performed using the Gel Shift Assay Systems (Promega). The oligonucleotide containing the NF-B consensus sequence probe was labeled with [-32P]ATP (Perkin Elmer Life Sciences Inc) using T4 polynucleotide kinase (Promega) and was purified by Sephadex G-25 chromatography (Roche Diagnostics Corp). Nuclear extracts (6 µg) from HeLa cells (controls) and noninfected and C pneumoniae–infected HAECs, prepared as previously described,27,29 were incubated at room temperature for 15 minutes with the Gel Shift Binding 5X Buffer provided by the manufacturer before the addition of the radiolabeled NF-B oligonucleotide probe. The protein-oligonucleotide complexes were then electrophoresed in a 4% nondenaturing polyacrylamide gel, and the radioactive bands were visualized by autoradiography.
C pneumoniae and the Protein Kinase Pathway
Confluent HAECs (3x106 cells) were incubated with C pneumoniae (1.5 IFU/cell) for 5, 15, 30, and 60 minutes at 37°C, 5% CO2, on a rocker platform. Noninfected cells and cells treated with phorbol, 12-myristate 13 acetate (PMA) (Calbiochem) were used as controls. After incubation, HAECs were washed with PBS/vanadate, and membrane and cytosolic fractions were separated as described.30 PKC activity was determined by immunoblotting using a purified rabbit polyclonal antibody raised against the PKC-specific peptide (AYQPYGKSVD), a sequence found in the C4 conserved region of PKC (Oncogenes Research Products). PKC isoforms (PKC-, -ß, -
, -
, -, -
, -
, -
, -
) were detected using a PKC sampler kit (BD Transduction Laboratories).
An expanded Materials and Methods section can be found in the online data supplement, available at http://www.circresaha.org.
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Results |
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Time-Dependent Upregulation of ICAM-1 Expression in C pneumoniae–Infected HAECs
As shown in Figure 1A, HAECs infected with C pneumoniae showed an increase in ICAM-1 protein expression 4 hours after infection with a peak at 12 hours after infection. High expression of ICAM-1 was still observed up to 24 hours after infection (Figure 1A). Heat and UV inactivation of C pneumoniae elementary bodies completely abolished the upregulation of ICAM-1 (Figure 1B).
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Figure 1C shows that ICAM-1 mRNA levels were not detectable in either noninfected control cells or in cells exposed to noninfected Hep-2 cells (mock cells). C pneumoniae–infected cells have considerably increased ICAM-1 mRNA levels compared with both noninfected and mock cells. The maximum increase in ICAM-1 mRNA levels was observed within 2 to 4 hours after the addition of C pneumoniae. The levels declined to near baseline levels after 8 hours of incubation. At 24 hours of incubation, ICAM-1 mRNA was no longer detected (Figure 1C).
Upregulation of ICAM-1 Expression in C pneumoniae–Infected HAECs Is Not Mediated by MAPK Activation
As shown in Figure 2A, increased phosphorylation of extracellular signal regulated kinase (ERK), mainly ERK1, was observed in HAECs infected by C pneumoniae. ERK activation was time dependent, peaking at 15 minutes after infection (Figure 2A). A slight activation of SAPK/JNK was also observed at 15 and 30 minutes after infection. Activation of p38 MAPK was not observed at any time point (Figure 2B).
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Inhibition of the MAPK pathway by MEK1/2 inhibitors (U0126 and PD98059) did not prevent upregulation of ICAM-1 expression in C pneumoniae–infected HAECs (Figures 3A and 3B), although, as expected, ERK phosphorylation was inhibited (Figure 3C). Inhibition of the JNK pathway using SP600125 also failed to inhibit the upregulation of ICAM-1 expression in C pneumoniae–infected HAECs (data not shown).
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Inhibition of NF-B Activation Completely Abolishes C pneumoniae–Induced ICAM-1 Expression by HAECs
As shown in Figure 4A, in HAECs infected by C pneumoniae, nuclear translocation of NF-B occurs in a time-dependent manner, with maximal activation occurring between 30 and 60 minutes after addition of C pneumoniae to the HAECs (Figure 4A). We have also shown, as described later in this section, that C pneumoniae induces NF-B activation by inducing phosphorylation of I-B (see Figure 6B).
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Pretreatment of HAECs for 1 hour with BAY117085 (BAY), a NF-B inhibitor, reduced the expression of ICAM-1 induced by C pneumoniae in a concentration-dependent manner, with complete inhibition at 10 mmol/L concentration (Figure 4B). These findings were confirmed in experiments using another NF-B inhibitor, CAPE, a specific inhibitor that prevents the translocation of the p65 subunit of NF-B to the nucleus and therefore inhibits its binding to the DNA. As shown in Figure 4C, activation of ICAM-1 by C pneumoniae was completely blocked by CAPE in a concentration-dependent manner. Addition of CAPE at the same concentrations to noninfected HAECs did not affect ICAM-1 protein (data not shown).
ICAM-1 Upregulation in C pneumoniae–Stimulated HAECs Is PKC Dependent
HAECs infected by C pneumoniae showed translocation of total PKC from the cytosol to the membrane as early as 30 minutes after addition of C pneumoniae. Significant decrease of PKC in the cytosolic fraction and corresponding increase in the membrane fraction was observed both at 30 and 60 minutes after infection (Figure 5A).
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Pretreatment of HAECs with calphostin C, which interacts with the common protein’s regulatory domain in all isozymes of PKC, prevented C pneumoniae–induced ICAM-1 upregulation in a concentration-dependent manner (Figure 5B). At lower concentrations (0.5 mmol/L), calphostin C prevented >50% of the ICAM-1 upregulation induced by C pneumoniae, but at higher concentrations (1 and 2 mmol/L), calphostin C not only prevented ICAM-1 upregulation but also decreased ICAM-1 expression below baseline levels (Figure 5B). Pretreatment of HAECs with bisindolylmaleimide I and indolocarbazole Gö 6976, which inhibit the classical and novel PKC isoforms, failed to prevent C pneumoniae–induced ICAM-1 expression. Similarly, pretreatment with PMA for 24 hours, which downregulates classical and novel PKC isoforms but not atypical PKCs, did not abolish ICAM-1 expression induced by C pneumoniae (data not shown).
PKC Mediates the Activation of NF-B in C pneumoniae–Infected HAECs
Because both PKC and NF-B pathways seem to regulate ICAM-1 expression, we examined the effect of PKC on NF-B activation. Figure 6A shows low levels of NF-B (p65) in the nuclear extracts from uninfected HAECs. Thirty to sixty minutes after HAECs were infected by C pneumoniae, a dramatic increase in NF-B (p65) was observed. NF-B (p65) was reduced by more than 80% with either calphostin C (PKC inhibitor) or BAY117085 (NF-B inhibitor). Experiments using the NF-B and the PKC inhibitors simultaneously were toxic for HAECs.
Furthermore, as shown in Figure 6B, calphostin C prevents C pneumoniae–induced phosphorylation of IB- in a concentration-dependent manner. C pneumoniae induces nuclear translocation of NF-B within 15 to 60 minutes (Figures 6A and 6C); in presence of calphostin C (1 and 2 mmol/L), complete inhibition of the C pneumoniae translocation of NF-B to the nucleus was observed (Figure 6C).
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Discussion |
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Endothelial dysfunction is not only the first step in the development of arteriosclerosis but also contributes to a critical late step, the formation of thrombi that may lead to vessel occlusion and acute cardiovascular events. Thus, it is not surprising that much effort has been focused on determining how infection of the endothelium by C pneumoniae causes endothelial dysfunction. Several mechanisms have been described; one of the best studied is the increased expression of ICAM-1. Our present data add to previously published data by demonstrating for the first time that C pneumoniae upregulates ICAM-1 expression by PKC-dependent activation of the NF-
B pathway. This study also shows that upregulation of ICAM-1 by C pneumoniae requires live bacteria, because heat-killed and UV-inactivated microorganisms did not induce ICAM-1 upregulation in HAECs. The effect of C pneumoniae in ICAM-1 upregulation is also specific of this microorganism, because mock-infected HAECs did not show a significant upregulation of ICAM-1 at mRNA or protein level. We have also demonstrated that although, as previously reported, C pneumoniae induces significant activation of the classical MAPK pathway in HAECs,10 the activation of this pathway is not responsible for the upregulation of ICAM-1 expression. Obviously, the signaling pathways that mediate upregulation of ICAM-1 differ according to cell type. In TNF-stimulated HUVECs, blocking ERK1/231 significantly reduced the expression of cell adhesion molecules. However, Chen et al7,32 found that although TNF and IL-1ß led to activation of ERK1/2, as well as upregulation of ICAM-1 expression in alveolar epithelial cells, blocking this pathway did not affect ICAM-1 expression. Similarly, in our studies, the borderline activation by C pneumoniae of the SAPK/JNK pathway was not responsible for the upregulation of ICAM-1 in HAECs. However, in Sertori cells, activation of the JNK/SAPK pathway led to upregulation of ICAM-1.33
It had been previously shown that NF-B elements within the proximal ICAM-1 promoter region mediate the increased expression of ICAM-1 in HUVECs and epithelial cells exposed to cytokines (TNF and IL-1ß), oxidized LDL, and bacterial lipopolysaccharide.32,34 We have now shown that C pneumoniae leads also to upregulation of ICAM-1 in HAECs through NF-B activation. Our results show that C pneumoniae stimulated translocation of NF-B in HAECs and that ICAM-1 expression was significantly reduced when HAECs were preincubated with specific NF-B inhibitors such as BAY117085, a novel compound shown to prevent cytokine-induced IB phosphorylation35 and, as a consequence, the cytokine-induced expression of cell adhesion molecules.35
Like ICAM-1 and other adhesion molecules (VCAM-1 and E-selectin), many other genes expressed in atheromatous plaques are regulated by NF-B,36 including those involved in chemotaxis (monocyte chemotactic protein 1) and thrombogenesis (tissue factor). More recently, translocation of NF-B after C pneumoniae infection was also reported in endothelial cells, smooth muscle cells, and human monocytic cell lines.23,37,38 Molestina et al39 demonstrated that in C pneumoniae–infected HUVECs, there is increased transcription of the monocyte chemotactic protein 1 via NF-B activation. Requirement for NF-B in the induction of tissue factor and PAI-1 expression by C pneumoniae has also been demonstrated in smooth muscle cells.23
Finally, we were able to demonstrate for the first time that PKC is activated in HAECs after their infection with C pneumoniae. Using 3 PKC inhibitors (calphostin C, bisindolymaleimide I, and indolocarbazole Gö 6976), we investigated the role of PKC in ICAM-1 upregulation. Each of the 3 inhibitors has unique structural characteristics and a unique mechanism of action. Calphostin C interacts with the common protein regulatory domain of all PKC isozymes and therefore inhibits all PKC isoenzymes. Our data showed that calphostin C inhibits ICAM-1 expression in C pneumoniae–stimulated HAECs in a concentration-dependent manner, thus indicating that ICAM-1 upregulation by HAECs is PKC dependent. However, neither bisindolymaleimide 1, which selectively inhibits the classical and, at higher concentrations, the novel isoforms of PKC, nor indolocarbazole Gö 6976, a highly selective inhibitor of the classical PKC isoforms, blocked the upregulation of ICAM-1 expression. Although PKC- and - were activated in HAECs stimulated with C pneumoniae (see the online data supplement), our studies with specific inhibitors seem to suggest that neither of these isoforms is involved in the upregulation of ICAM-1. Thus, it is likely that C pneumoniae induces ICAM-1 upregulation in HAECs via other novel or atypical PKC isoforms.27 Rahman et al,27 using a dominant-negative approach, were able to demonstrate in TNF-stimulated endothelial cells that the atypical isoform PKC
was able to prevent IB degradation and NF-B binding to the ICAM-1 promoter.27 We were unable to confirm the involvement of other PKC isoforms because of the unavailability of specific inhibitors.
However, based on the above data and on some experimental evidence suggesting that PKC activation leads to the dissociation of the NF-B/IkB complex and therefore to the translocation of NF-B to the nucleus and activation of its target genes,40 we investigated the possible effect of PKC activation by C pneumoniae on the NF-B inhibitor, I-B. Inhibition of the PKC pathway in C pneumoniae–infected HAECs by calphostin C blocked ICAM-1 upregulation by preventing I-B phosphorylation and, as a consequence, the translocation of NF-B to the nucleus. The degree of inhibition of NF-B by calphostin C was similar to that induced by the specific NF-B inhibitor BAY117085. A similar pathway regulates the events that modulate adhesion and filtration of leukocytes and upregulation of ICAM-1 expression on HUVECs and mesangial cells exposed to high levels of glucose.37,41 Hence, in C pneumoniae–infected HAECs, we demonstrate that PKC enhances NF-B activity by direct phosphorylation of its cytoplasmic inhibitor, I-B, thus facilitating NF-B binding to the NF-B motif in the ICAM-1 promoter.
In conclusion, the present study shows for the first time that PKC is activated in HAECs on infection with C pneumoniae. Activation of PKC leads to NF-B activation, and that, in turn, leads to increased transcription of the ICAM-1 gene. Experiments designed to additionally elucidate the mechanism of selective induction of ICAM-1 signaling pathways by C pneumoniae and the role of different cell lines in this process are presently ongoing in our laboratory. C pneumoniae–induced activation of ICAM-1 is likely to contribute to the chronic inflammatory events associated with arteriosclerosis.
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Acknowledgments |
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This research was supported in part by the Research Service of the Ralph H. Johnson Department of Veterans Affairs Medical Center (to M.F.L.-V.) and by grant HL-46815 from the NIH (to M.F.L.-V.). The authors also acknowledge the contribution of Jeffrey Greve, PhD, Director of Molecular Technologies, Bayer Pharmaceutical Division, Berkeley, Calif, who generously provided us with ICAM-1 cDNA.
Received November 12, 2002; revision received March 24, 2003; accepted April 16, 2003.
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