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*
Department of Internal Medicine, Justus-Liebig-University, Giessen, Germany;
Institute of Medical Microbiology, Justus-Liebig-University, Giessen, Germany;
Institute of Microbiology, Heinrich-Heine-University, Düsseldorf, Germany; and
§
Charite, Department of Internal Medicine, Humboldt-University, Berlin, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation/translocation
occurred within 10–15 min. Increased mRNA and surface expression of
E-selectin, ICAM-1, and VCAM-1 were noted within hours. Thus, C.
pneumoniae triggers a cascade of events that could lead to
endothelial activation, inflammation, and thrombosis, which in turn may
result in or may promote atherosclerosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The role of C. pneumoniae in atheroma formation has not been
studied in detail. Monocytes, macrophages, and smooth muscle cells have
been shown to be susceptible for C. pneumoniae infection
(9, 10, 11). Chlamydiae may reside and replicate in
monocytes/macrophages and induce a chronic immune activation resulting
in TNF-
, IL-1ß, IL-6, and IFN-
release as well as up-regulation
of CD14 (12, 13). Little is known about C.
pneumoniae-induced endothelial cell alterations and C.
pneumoniae-mediated interactions among all cell types involved in
the progress of atherosclerosis.
Airway-derived organisms may be able to spread systemically via at least two different ways: 1) carried within monocytes following pulmonary infection or 2) by direct access to the blood stream causing a short interval of chlamydial bacteremia. Chronic monocyte/macrophage infection as well as direct activation of endothelial cells may contribute to local inflammation by inducing cytokine release and increased expression of adhesion molecules, which in turn may result in enhanced rolling, adhesion, and transendothelial migration of leukocytes.
Adhesion of circulating leukocytes to endothelial cells is an early step in an inflammatory reaction that is regulated by a complex communication between the cell types involved. Recent studies revealed that multiple receptor-ligand pairs act sequentially and in an overlapping manner to effect initial attachment, rolling, firm adhesion, and finally transmigration of leukocytes (14, 15, 16). For leukocyte adhesion to activated endothelial cells, separate receptor-ligand pairs are involved: ICAM-1 and ß2 integrins (CD11a/CD18, CD11b/CD18), VCAM-1 and very late Ag-4 (VLA4),3 P-selectin and P-selectin-glycoprotein ligand-1, E-selectin and E-selectin ligand-1, as well as sialyl Lewisx and related carbohydrates on leukocytes (14, 17, 18, 19).
Therefore, the first objective of the present study was to assess C. pneumonia’s capability to infect and activate HUVEC and human aortic endothelial cells (HAEC). Enhanced expression of endothelial adhesion molecules was accompanied by an increased rolling, adhesion, and transmigration of polymorphonuclear leukocytes (PMN) and monocytes. These effects were reduced by mAbs directed against different adhesion molecules on leukocytes and/or endothelial cells.
There is limited knowledge of the mechanisms of C. pneumoniae entry into endothelial cells. The chlamydial growth cycle is initiated when an infectious elementary body (EB) attaches to a susceptible target cell, promoting entry into a host cell-derived phagocytic vesicle. EB develop into reticular bodies, a process that could be detected metabolically within 15 min and microscopically 12–15 h after addition of Chlamydiae to HEp-2 and Hela-229 cells (20, 21). The length of the complete developmental cycle, as studied in cell culture models, is 48–72 h (22, 23, 24).
Almost nothing is known about the signal transduction pathways
initiated in the host cell upon Chlamydiae-target cell
interaction (intracellular Ca2+-increase, activation of
protein and tyrosine kinases, mitogen-activated protein kinases (MAPK),
NF-B translocation). Therefore, the second objective of the present
study was to assess C. pneumoniae’s capability to activate
host cell signal transduction pathways. C. pneumoniae
induced an immediate increase of total protein tyrosine phosphorylation
in HUVEC and HAEC, especially of phosphorylated p42/p44 MAPK and an
activation/translocation of endothelial cell NF-B with subsequently
increased transcription and translation of E-selectin, ICAM-1, and
VCAM-1.
Overall, the data presented indicate that C. pneumoniae can infect and activate human endothelial cells and suggest that C. pneumoniae-induced endothelial cell alterations may promote atherosclerosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tissue culture plasticware was obtained from Becton Dickinson (Heidelberg, Germany). MCDB131 medium, PBS, trypsin-EDTA solution, HEPES, and FCS were from Life Technologies (Karlsruhe, Germany). Collagenase (CLS type II) was purchased from Worthington Biochemical (Freehold, NJ). Percoll and Ficoll-Paque were obtained from Pharmacia (Uppsala, Sweden). All other reagents were obtained from Sigma (Munich, Germany).
Monoclonal Abs
Purified freeze-dried mAb directed against CD11a (25.3.1), CD11c (BU15), CD18 (7E4), CD29 (Lia 1/2), CD31 (Hec-7), CD49d (HP2/1), and P-selectin (CLB/thromb6) were obtained from Coulter/Immunotech (Marseille, France); mAb directed against CD11b (clone 44), E-selectin (1.2B6), ICAM-1 (15.2), and VCAM-1 (1.G11B1) were obtained from Dianova (Hamburg, Germany).
Cell culture
Preparation of HUVEC. Cells were isolated from umbilical cord veins and identified according to the method of Jaffe et al. (25). Briefly, cells obtained from collagenase digestion were washed, resuspended in MCDB131/5% FCS, and seeded into tissue culture flasks (80 cm2), well plates, or glass coverslips (26, 27).
HAEC. HAEC were obtained from Clonetics (San Diego, CA). HAEC were grown to confluence using EGM-2/2% FCS (Clonetics) in tissue culture flasks (80 cm2), well plates, or glass coverslips.
Isolation of human PMN. Heparinized human donor blood was centrifuged in a discontinuous Percoll gradient to yield a PMN fraction of >97% purity as described (28, 29).
Isolation and labeling of human monocytes. Human monocytes were isolated from anti-coagulated whole blood or from buffy coats by centrifugation on Ficoll-Paque density gradient followed by counterflow centrifugation elutriation in a Beckman J6-MC centrifuge using a JE-6 elutriation rotor and a 5.5-ml Sanderson chamber (30). Monocyte suspension was 90 ± 4% (n = 20) pure with 7% lymphocytes and <2% granulocytes as determined by light scatter (FACScan; Becton Dickinson).
Chlamydial strain
C. pneumoniae strain GiD was used. This strain was originally isolated from a patient with bronchitis. The isolate was identified to be C. pneumoniae by staining inclusions formed in cell culture with a species-specific mAb and by sequence analysis of the entire omp1 gene (31).
C. pneumoniae GiD was grown to high titers in cyclohexamide-treated HEp-2 cells (20). Infected monolayers were harvested from culture flasks and sonicated for 30 s. Cellular debris was removed by centrifugation at 500 x g for 10 min at 4°C. Aliquots diluted with an equal volume of sucrose-phosphate-glutamate buffer supplemented with 10% FCS were stored at -75°C until use. Titration in cyclohexamide-treated Hep-2 cells was performed with a thawed aliquot (in triplicate). Chlamydiae concentrations used were expressed as inclusion forming units (IFU)/ml.
C. pneumoniae infection assay
C. pneumoniae suspensions were thawed, diluted in MCDB131 medium, and inoculated onto HUVEC or HAEC grown in 24- or 96-well tissue culture plates. Plates were centrifuged at 800 x g for 1 h at 35°C. After incubation at 37°C for 1 h, the supernatant was replaced by fresh medium and plates were processed for additional experiments at the times indicated in the figure legends.
Cell surface ELISA for P-selectin, E-selectin, ICAM-1, and VCAM-1-expression
Expression of different adhesion molecules on monolayer, of endothelial cells preincubated with C. pneumoniae was determined by cell surface ELISA as described previously (32).
Leukocyte rolling and adhesion assay
Leukocyte rolling and adhesion was determined using a parallel plate flow chamber according to Lawrence and Springer (33). Confluent endothelial monolayer were preincubated with C. pneumoniae. A suspension of 106 leukocytes/ml was perfused through the chamber at a constant wall shear stress of 1.0 dyne/cm2 (syringe pump sp100i; World Precision Instruments, Sarasota, FL). Interactions were visualized using a phase contrast video microscope (IMT-2; Olympus Optical, Hamburg, Germany, with a KP-C551 CCD camera; Hitachi, Rodgau, Germany) and videotaped (JVC HR-S7000; JVC, Friedberg, Germany) the entire time period of leukocyte perfusion. Rolling in the parallel plate flow chamber was measured in five high power fields for each experiment. Images were recorded at real time and played back at six- or ninefold slower speed. The tape was paused to mark the location of cells, and the displacement of the center of individual cells was measured 2–4 s later. "Rolling" was expressed as the number of rolling cells/high power field (20x objective) during a 3-min observation period. Leukocytes were considered to be adherent after 30 s of stable contact with the monolayer. Adhesion was determined after 5 min of perfusion by analysis of 10 random high power fields (20x) from videotape (33). Results are expressed as rolling or adherent cells/high power field.
Leukocyte transmigration assay
For transmigration assays, HUVEC were grown on polycarbonat-membranes (6.5-mm Trans-well cell-culture inserts, 8-µm pore size; Corning-Costar, Cambridge, MA). Then, 105 2'7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethylester-labeled leukocytes (1 µM, 15 min) were added to the upper chamber of the C. pneumoniae-preincubated HUVEC monolayer and incubated for 2 h at 37°C. Fluorescence intensity in the lower compartment was quantitated with a FluoroMax2/MicroMax (Instruments S.A., Munich, Germany) and represented migrated leukocytes.
Western blot analysis
For detection of tyrosine phosphorylation HUVEC or HAEC grown on six-well culture plates were stimulated with 6.5 x 104 IFU/ml C. pneumoniae. Cell proteins (40 µg/lane) were separated by SDS-PAGE according to Laemmli et al. (34), blotted on Hybond enhanced chemiluminescence membranes (Amersham, Dreieich, Germany), blocked, incubated with rabbit-mAb against tyrosine-phosphorylated proteins (RC20; Transduction Laboratories, Lexington, Kentucky) or phosphorylated p42/p44 MAPK (New England Biolabs, Beverly, MA), and detected by enhanced chemiluminescence (Amersham).
Electrophoretic mobility shift assay (EMSA)
After stimulation with 6.5 x 104 IFU/ml
C. pneumoniae for different time points, nuclear protein was
isolated as described by Newton et al. (35). The consensus NF-B
oligonucleotide (5'-AGT TGA GGG GAC TTT CCC AGG-3') (Promega, Mannheim,
Germany) was end-labeled with [32P]ATP using T4
polynucleotide kinase (Bioline, Berlin, Germany). Unincorporated
nucleotides were separated on a Sephadex G-25 spun column (Pharmacia,
Freiburg, Germany). EMSA binding reactions were performed by first
preincubating 5 µg of nuclear extract with 1 µg of poly(dI-dC) in
binding buffer (10 mM Tris, pH 7.7, 50 mM NaCl, 20% glycerol, 1 mM
DTT, 0.5 mM EDTA) for 20 min. Approximately 10,000 cpm (0.2 ng) of
32P-labeled DNA probe was then added and allowed to bind
for 20 min. The reaction mixture was subjected to electrophoresis on
7% native acrylamide gels before vacuum drying and exposing to a
storage phosphor screen for quantification and documentation
(PhosporImager, Molecular Dynamics, Sunnyvale, CA). Competition
experiments were performed as above except that 100-fold excess
unlabeled competitor DNA was added to the incubations.
Immunofluorescence analysis of NF-B nuclear translocation
After stimulation of HUVEC grown on glass chamber slides (Falcon
CultureSlide, Becton Dickinson, Rutherford, NJ) with 6.5 x 104
IFU/ml C. pneumoniae for 30 min, cells were fixed and
permeabilized with acetone/methanol (-20°C, 50:50 v/v) for 5 min.
Human Ig was used to reduce nonspecific binding, and the primary Ab
(polyclonal rabbit anti-human NF-B p65 Ab; Santa Cruz
Biotechnology, Santa Cruz, CA) was added for 30 min. Thereafter, cells
were washed thrice and exposed to an ALEXA-488-conjugated goat
anti-rabbit Ig Ab (Molecular Probes, Eugene, OR) for 15 min. After
washing thrice with PBS, coverslips were sealed and examined in a
Olympus IMT-2 fluorescence microscope (Olympus Optical, equipped with
an Olympus OM-4 camera) with an Olympus 60x objective.
NF-B reporter gene assay
Six NF-B DNA binding sites (5'-GGG GAC TTT CCC
T-3'; italics indicates original binding site) were inserted
into the SmaI site in a pGL3basic vector (Promega).
Downstream of this six NF-B binding region, a minimal ß-globin
promoter (containing a TATA box) was inserted into the
XhoI/HindIII sites followed by the luciferase
gene (pGL3.BG.6B). HUVEC were transiently transfected with 2
µg NF-B plasmid using SuperFect transfection reagent (Qiagen,
Hilden, Germany). Transfected HUVEC were stimulated, harvested in
reporter lysis buffer (Promega), and total protein was measured using
the Bio-Rad reagent (Munich, Germany). Luciferase-assay was performed
using a commercial kit (Promega). Luminescence was measured in a Lumat
LB 9501 luminometer (Berthold, Wildbad, Germany). Relative luminescence
readings were normalized to total protein and expressed as fold
activation relative to control ± SEM. A control plasmid was
created by inserting six mutated NK-B sites (5'-GGC CAC
TTT CCC T-3'; italics indicates changes/mutation compared to the
nonmutated binding site) into the same vector
(pGL3.BG.6B.mut)
Northern blot analysis
RNA was extracted using the guanidinium isothiocyanate method as
described by Chomczynski and Sacchi (36). cDNA probes were
labeled with [-32P]dCTP (>3000 Ci/mmol) by random
priming (Rediprime DNA labeling system; Amersham, Braunschweig,
Germany), added to the prehybridization chambers, and incubated for
12–16 h at 42°C. E-selectin and VCAM-1 cDNA probes were a friendly
gift of Dr. D. Simmons (Imperial Cancer Research Fund, Institute of
Molecular Medicine, Oxford, U.K.), and the ICAM-1 probe was kindly
provided by Dr. D. Vestweber (Department of Cell Biology,
University of Münster, Münster, Germany). The
598-bp cDNA fragment of GAPDH was obtained as previously
described (37).
Statistical methods
Depending on the number of groups (A) and the number of
different time points studied (B), data of Figs. 1
b and 2,
a, c, and e were analyzed by a two-way
ANOVA. An one-way ANOVA was used for data of Figs. 1, a and
c, 2, b, d, and f, 3,
a–c, and 4b. Values of p < 0.05
were considered significant (38).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C. pneumoniae has been demonstrated to infect and thereby activate human endothelial cells (10, 11, 39). To investigate leukocyte-endothelial cell interaction, we infected HUVEC with the recently described C. pneumoniae strain GiD (31). GiD turned out to be a highly effective Chlamydia strain and was able to replicate and to form inclusions in HUVEC with a mean titer of 2.6 x 103 IFU/ml as compared with growth in cyclohexamide-treated HEp-2 cells at a mean titer of 6.5 x 104 IFU/ml. Inclusions were visualized using a FITC-conjugated genus-specific mAb (Chlamydia culture confirmation system; Sanofi Diagnostics Pasteur, Freiburg, Germany) (data not shown).
C. pneumoniae-mediated leukocyte endothelial cell interaction
To investigate C. pneumoniae-mediated leukocyte-endothelial cell interaction, HUVEC were stimulated with different concentrations of strain GiD. Intracellular infection resulted in a profound cell activation with subsequently enhanced leukocyte rolling, adhesion, and transmigration. Inactivation of bacterial LPS by pretreatment of C. pneumoniae with polymyxin B (10 µg/ml) had no effect on leukocyte-HUVEC interaction. Killing of C. pneumoniae by heating (90°C, 60 min) reduced chlamydial-mediated effects almost completely (data not shown).
Monocyte rolling on the C. pneumoniae-infected endothelial
cell monolayer was significantly increased after 4 h and 24 h
(Fig. 1
a). Leukocyte rolling
after 4 h was reduced by a mAb against endothelial E-selectin
(56%). mAb directed against CD11b/18 or CD49d on monocytes and VCAM-1
on endothelial cells were ineffective at that time point. After 24
h of C. pneumoniae infection, preincubation of endothelial
cells with anti-VCAM-1 as well as monocytes with anti-CD49d
diminished leukocyte rolling by 55 and 70%, respectively,
whereas Abs directed against CD11b/18 or E-selectin were ineffective.
Furthermore, intracellular infection of HUVEC dose- and
time-dependently increased leukocyte adhesion to endothelial cells with
maximal effects seen at the concentration of 6.5 x
104 IFU/ml. Adhesion increased after 2 h and remained
elevated up to 72 h (Fig. 1b, left,
monocytes; right, PMN). After 4 h of HUVEC incubation
with C. pneumoniae, PMN and monocyte adherence was reduced
by anti-E-selectin (45.5 and 58.3%) and anti-ICAM-1 Abs (42.8
and 63.2%) (Table I). Monocyte adhesion
was also diminished by an anti-VCAM-1 mAb (28.5%).
Anti-ß1 (VLA4) and ß2 integrin
Abs reduced leukocyte adhesion to C. pneumoniae-stimulated
endothelial cells by up to 90% (see Table I for details).
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). Monocyte adhesion to C.
pneumoniae-infected HUVEC was followed by a subsequent dose- and
time-dependently enhanced transendothelial migration (Fig. 1c). This parameter showed a similar inhibitory profile with
respect to the different mAb used (for details, see Table II).
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Cell surface ELISA for endothelial adhesion molecule expression
was performed to further characterize C. pneumoniae-mediated
leukocyte-endothelial cell interaction. Infection of HUVEC and HAEC
with C. pneumoniae dose- and time-dependently increased
expression of E-selectin, ICAM-1, and VCAM-1 (Fig. 2) on endothelial cells (HUVEC and HAEC).
Maximal effects in this study occurred in the presence of 6.5 x
104 IFU/ml (HAEC, data not shown). E-selectin expression in
C. pneumoniae-stimulated endothelial cells increased 2
h postinfection, peaked at 4 h, and declined to almost baseline
after 18–24 h. Even the lowest Chlamydia concentration
tested was able to induce a significant E-selectin response (Fig. 2a). ICAM-1 and VCAM-1 surface expression in C.
pneumoniae stimulated cells increased 4–8 h postinfection, peaked
at 12–24 h (HUVEC) and persisted up to 72 h (Fig. 2, c
and e). Compared with HUVEC, the kinetics of the ICAM-1 and
VCAM-1 response in HAEC were delayed, and absolute levels of protein
expression diminished.
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Northern blot analysis was performed to verify mRNA up-regulation
for E-selectin, ICAM-1, and VCAM-1 (Fig. 3). E-selectin mRNA peaked at 2 h of
C. pneumoniae stimulation and almost completely disappeared
after 8 h (Fig. 3a). ICAM-1 and VCAM-1 mRNA peaked at
2 h, declined to almost baseline after 8 h, and was
detectable again after 24 h (Fig. 3, b and
c).
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B in C. pneumoniae-mediated
endothelial cell activation
Previous studies have elaborated the importance of NF-B
for the regulation of the transcriptional activities of the E-selectin,
ICAM-1, and VCAM-1 genes (40). Multiple NF-B binding sites
have been located in the promoters of all three genes (41, 42, 43).
NF-B activation in C. pneumoniae-infected endothelial
cells was demonstrated by the enhanced binding-capacity of NF-B to
corresponding consensus oligonucleotides using band shift assays (Fig. 4a). This point was verified by
immunofluorescence, which indicated increased NF-B translocation
within 15–30 min of C. pneumoniae stimulation with
maximal effects after 30 min (Fig. 4c).
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B activation was confirmed by enhanced NF-B-dependent
transcription of a luciferase-gene transiently transfected into HUVEC
(Fig. 4b). HUVEC were transiently transfected for 2 h
with 2 µg NF-B-luciferase plasmid (pGL3.BG.6B), stimulated, and
harvested in reporter lysis buffer after 4 h. NF-B activity
(indicated as chemiluminescence/µg protein) increased
dose-dependently in C. pneumoniae-stimulated endothelial
cells. Chlamydia-stimulated HUVEC, which had been
transiently transfected in parallel with the mutated
NF-B-luciferase plasmid (pGL3.BG.6B.mut), revealed no detectable
luciferase activity (data not shown). C. pneumoniae induced rapid total protein tyrosine and p42/p44 MAPK phosphorylation in endothelial cells
Activation of p42/p44 MAPK isoforms is an early step in cell
activation and intracellular signaling. Western blot analysis showed a
time-dependently enhanced total protein tyrosine phosphorylation (Fig. 5a), specifically of phosphorylated p42
and p44 MAPK isoforms in HUVEC (Fig. 5b) and HAEC (Fig. 5c). p42/p44 MAPK-phosphorylation peaked at 10 (HUVEC) to 15
min (HAEC).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In vivo, endothelial cell infection may occur directly via blood-borne C. pneumoniae or indirectly as shown by recent work by Gaydos et al. demonstrating that C. pneumoniae transfer to human endothelial cells may proceed by cell-to-cell spread from infected adherent mononuclear phagocytes (44). Moreover, mice intranasally infected with C. pneumoniae showed evidence of systemic chlamydial dissemination via macrophages (45).
Almost nothing is known about the signal transduction pathways
activated upon target cell infection. Our studies, aimed to identify
possible intracellular signaling steps involved, indicated that protein
tyrosine phosphorylation, MAPK activation, and NF-B
activation/translocation occurred within 10–15 min of
Chlamydia addition to endothelial cells. Subsequently
increased mRNA levels and translation of E-selectin, ICAM-1, and VCAM-1
were noted. These results indicate that C. pneumoniae
triggers immediate events of cell activation, suggesting that C.
pneumoniae attachment is sufficient to initiate an endothelial
response and that bacterial uptake may not be required for this
process. Chlamydia, pretreated with polymyxin B to
inactivate LPS, were still able to stimulate endothelial cells, while
heat-killed Chlamydia did not induce adhesion molecule
expression, suggesting that living bacteria but not chlamydial LPS may
be required for cell activation.
After initial attachment, Chlamydiae are internalized; they dissociate themselves from the endocytotic pathway by actively modifying the vacuole to become fusogenic with exocytic vesicles (46). Interaction with this secretory pathway appears to provide a pathogenic mechanism that allows chlamydiae to establish themselves in a site that is not destined to fuse with lysosomes. Further studies are required to determine the relationship between distinct steps of this chlamydial development cycle and initiation of host cell signaling pathways.
Upon infection with intracellular pathogens, NF-B activation,
enhanced expression of adhesion molecules, and increase in
leukocytes-target cell interaction may be an uniform and nonspecific
endothelial cell reaction. Actually, we recently reported a similar
pattern of host cell activation in Listeria
monocytogenes-infected endothelial cells (32). In subsequent
studies, we were able to identify Listeria-induced host
cell-derived ceramide as the second messenger that initiated NF-B
activation and that therefore represented the link between
Listeria infection and endothelial cell activation (47).
However, preliminary data in C. pneumoniae-infected
endothelial cells indicate that ceramides are not increased, suggesting
that, in case of Chlamydia, they are not a key intracellular
mediator (Krüll et al., unpublished observation). Overall, we
believe that bacteria-induced target cell activation results from
stimulation of common and distinct (dependent on pathogen-specific
virulence factors) signaling pathways in host cells.
The interpretation of our study is limited to cultured human large vessel endothelial cells. For an exact analysis of C. pneumoniae-related alterations of endothelial function in clinical disorders, it would be desirable to also study human small vessel endothelial cells of different organs. However, the isolation and culture of these cells in sufficient quantities is difficult, and therefore the applicability of the data presented to human disease must be verified in further studies. At least with respect to large vessel endothelium, we provided data demonstrating no substantial difference between cultured human large vein and aortic endothelial cells.
In conclusion, we present evidence that C. pneumoniae
can infect HUVEC and HAEC and activate different signal transduction
pathways: protein tyrosine phosphorylation, MAPK stimulation, and
activation/translocation of NF-B occurred within minutes. Within
hours, increased mRNA and surface expression of E-selectin, ICAM-1, and
VCAM-1 was noted, which in turn resulted in enhanced leukocyte
(monocytes, PMN)-HUVEC interaction. Overall, the data presented suggest
that C. pneumoniae infection triggers a cascade of events
that could lead to endothelial damage, inflammation, and thrombosis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Norbert Suttorp, Charite, Department of Internal Medicine/Infectious Diseases Humboldt-University, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail address:
3 Abbreviations used in this paper: VLA, very late Ag; HAEC, human aortic endothelial cells; PMN, polymorphonuclear leukocytes; MAPK, mitogen activated protein kinase; IFU, inclusion forming unit; EB, elementary body; EMSA, electrophoretic mobility shift assay.
Received for publication June 29, 1998. Accepted for publication January 4, 1999.
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References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-toxin potently induce neutrophil adhesion to cultured human endothelial cells. J. Immunol. 157:4133.[Abstract]
B by actinomycin D and cycloheximide in epithelial cells. Biochem. Biophys. Res. Commun. 218:518.[Medline]
B. Agents Actions Suppl. 47:135.[Medline]
B activation, and E-selectin expression in human endothelial cells. J. Immunol. 161:3010.This article has been cited by other articles:
|
|
 |
|
  F. N. Wuppermann, K. Molleken, M. Julien, C. A. Jantos, and J. H. Hegemann Chlamydia pneumoniae GroEL1 Protein Is Cell Surface Associated and Required for Infection of HEp-2 Cells J. Bacteriol., May 15, 2008; 190(10): 3757 - 3767. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Schmeck, W. Beermann, P. D. N'Guessan, A. C. Hocke, B. Opitz, J. Eitel, Q. T. Dinh, M. Witzenrath, M. Krull, N. Suttorp, et al. Simvastatin Reduces Chlamydophila pneumoniae-Mediated Histone Modifications and Gene Expression in Cultured Human Endothelial Cells Circ. Res., April 25, 2008; 102(8): 888 - 895. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-J. Jiang, C.-C. Kuo, M. W. Berry, A. W. Lee, and L. A. Campbell Identification and Characterization of Chlamydia pneumoniae-Specific Proteins That Activate Tumor Necrosis Factor Alpha Production in RAW 264.7 Murine Macrophages Infect. Immun., April 1, 2008; 76(4): 1558 - 1564. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Takaoka, L. A. Campbell, A. Lee, M. E. Rosenfeld, and C.-C. Kuo Chlamydia pneumoniae Infection Increases Adherence of Mouse Macrophages to Mouse Endothelial Cells In Vitro and to Aortas Ex Vivo Infect. Immun., February 1, 2008; 76(2): 510 - 514. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Droemann, J. Rupp, T. Goldmann, U. Uhlig, D. Branscheid, E. Vollmer, P. Kujath, P. Zabel, and K. Dalhoff Disparate Innate Immune Responses to Persistent and Acute Chlamydia pneumoniae Infection in Chronic Obstructive Pulmonary Disease Am. J. Respir. Crit. Care Med., April 15, 2007; 175(8): 791 - 797. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Liu, J.-f. Wu, Y.-y. Zhan, H.-z. Chen, X.-y. Zhang, and Q. Wu Regulation of the Orphan Receptor TR3 Nuclear Functions by c-Jun N Terminal Kinase Phosphorylation Endocrinology, January 1, 2007; 148(1): 34 - 44. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Harokopakis, M. H. Albzreh, M. H. Martin, and G. Hajishengallis TLR2 Transmodulates Monocyte Adhesion and Transmigration via Rac1- and PI3K-Mediated Inside-Out Signaling in Response to Porphyromonas gingivalis Fimbriae. J. Immunol., June 15, 2006; 176(12): 7645 - 7656. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. D. N'Guessan, S. Hippenstiel, M. O. Etouem, J. Zahlten, W. Beermann, D. Lindner, B. Opitz, M. Witzenrath, S. Rosseau, N. Suttorp, et al. Streptococcus pneumoniae induced p38 MAPK- and NF-{kappa}B-dependent COX-2 expression in human lung epithelium Am J Physiol Lung Cell Mol Physiol, June 1, 2006; 290(6): L1131 - L1138. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Schmeck, S. Huber, K. Moog, J. Zahlten, A. C. Hocke, B. Opitz, S. Hammerschmidt, T. J. Mitchell, M. Kracht, S. Rosseau, et al. Pneumococci induced TLR- and Rac1-dependent NF-{kappa}B-recruitment to the IL-8 promoter in lung epithelial cells Am J Physiol Lung Cell Mol Physiol, April 1, 2006; 290(4): L730 - L737. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Krull, P. Bockstaller, F. N. Wuppermann, A. C. Klucken, J. Muhling, B. Schmeck, J. Seybold, C. Walter, M. Maass, S. Rosseau, et al. Mechanisms of Chlamydophila pneumoniae-Mediated GM-CSF Release in Human Bronchial Epithelial Cells Am. J. Respir. Cell Mol. Biol., March 1, 2006; 34(3): 375 - 382. [Abstract] [Full Text] [PDF]
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 |
|
 Q. Chen, P. R. Stone, L. M.E. McCowan, and L. W. Chamley Phagocytosis of Necrotic but Not Apoptotic Trophoblasts Induces Endothelial Cell Activation Hypertension, January 1, 2006; 47(1): 116 - 121. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-H. Chou, H. Yumoto, M. Davey, Y. Takahashi, T. Miyamoto, F. C. Gibson III, and C. A. Genco Porphyromonas gingivalis Fimbria-Dependent Activation of Inflammatory Genes in Human Aortic Endothelial Cells Infect. Immun., September 1, 2005; 73(9): 5367 - 5378. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Pollmann, M. Nordhoff, A. Pospischil, K. Tedin, and L. H. Wieler Effects of a Probiotic Strain of Enterococcus faecium on the Rate of Natural Chlamydia Infection in Swine Infect. Immun., July 1, 2005; 73(7): 4346 - 4353. [Abstract] [Full Text] [PDF]
|
|
|
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|
|
B. Opitz, S. Forster, A. C. Hocke, M. Maass, B. Schmeck, S. Hippenstiel, N. Suttorp, and M. Krull Nod1-Mediated Endothelial Cell Activation by Chlamydophila pneumoniae Circ. Res., February 18, 2005; 96(3): 319 - 326. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Schmeck, J. Zahlten, K. Moog, V. van Laak, S. Huber, A. C. Hocke, B. Opitz, E. Hoffmann, M. Kracht, J. Zerrahn, et al. Streptococcus pneumoniae-induced p38 MAPK-dependent Phosphorylation of RelA at the Interleukin-8 Promotor J. Biol. Chem., December 17, 2004; 279(51): 53241 - 53247. [Abstract] [Full Text] [PDF]
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