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Infect Immun, April 1998, p. 1370-1376, Vol. 66, No. 4
Division of Infectious Diseases,
Received 19 November 1997/Returned for modification 5 January
1998/Accepted 15 January 1998
Chlamydia pneumoniae is a respiratory pathogen that has
been associated with chronic inflammatory diseases such as asthma and
atherosclerosis. Recent isolation of C. pneumoniae from
human carotid and coronary atheromas provides additional support for a
role of this organism in atherogenesis. We characterized the coronary
strain C. pneumoniae A-03 by sequence analysis of the major
outer membrane protein gene (omp1). In addition, the in vitro activities of A-03 and three respiratory strains of C. pneumoniae (BAL-16, TW-183, and T-2634) were examined in infected
human umbilical vein endothelial cells (HUVEC) by analysis of the
production of interleukin-8 (IL-8), monocyte chemotactic protein 1 (MCP-1), and soluble intercellular cell adhesion molecule 1 (sICAM-1). Sequence analysis of omp1 of C. pneumoniae
A-03, compared to prototype strains TW-183 and AR-39, revealed five
nucleotide changes resulting in nonsynonymous codons. Of interest was a
nonconservative amino acid substitution (Ser to Pro) in position 61 of
variable segment 1. In vitro, the extent of MCP-1, IL-8, and sICAM-1
production was dependent on the C. pneumoniae strain
examined at low multiplicities of infection following 24 h of
incubation. Strain A-03 displayed the lowest stimulatory activity in
infected HUVEC, while T-2634 induced the highest levels of MCP-1, IL-8,
and sICAM-1 among all strains examined. Heat-inactivated C. pneumoniae failed to stimulate production of these proteins by
all strains tested. In contrast, only partial inhibition was observed
by UV-inactivated organisms. Results from this study demonstrate that
unlike prototype respiratory strains of C. pneumoniae, the
coronary strain A-03 displays divergence in the omp1 gene.
In addition, the stimulation of chemokines and adhesion molecules
involved in the recruitment of leukocytes to sites of inflammation by
C. pneumoniae may be important in the pathogenesis of
diseases associated with this organism, including atherosclerosis.
Chlamydia pneumoniae is a
respiratory pathogen that causes sinusitis, bronchitis, pneumonia, and
other acute respiratory infections (12, 13, 23). Infection
with this organism has also been associated with chronic inflammatory
diseases such as asthma (14) and atherosclerosis (24,
26, 29, 32). The role of C. pneumoniae in
atherosclerosis remains unclear despite the detection of this bacterium
in atheromas by numerous studies (21, 22, 33), including a
report from our laboratory documenting the isolation of C. pneumoniae from the coronary artery of a patient with severe coronary atherosclerosis (30). Isolation of this organism
from a carotid artery atheroma has been reported as well
(17).
The pathologic significance of C. pneumoniae in chronic
inflammatory diseases is not well understood. This organism is capable of remaining viable in the host despite antibiotic treatment of chronic
respiratory infections following acute illness (15). One of
the hallmarks of diseases such as asthma and atherosclerosis includes
the accumulation of blood-borne leukocytes into the inflamed tissues in
response to antigenic stimuli. This process is initiated with the
binding of leukocytes to activated endothelium via induced expression
of adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1) and leukocyte function antigen 1 (27). Leukocyte chemotaxis and migration across the endothelium are modulated by
numerous chemokines (i.e., chemotactic cytokines), including interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1), which
have specificities for neutrophils and monocytes, respectively (2). The fact that these chemokines have been detected in
human atheromas suggests that they may play important pathophysiologic roles in atherogenesis (28, 36).
The in vivo effects of C. pneumoniae on the production of
inflammatory mediators have not been studied in detail. Lung pathology in mice models is characterized by patchy interstitial pneumonitis, with polymorphonuclear leukocyte infiltration in the early stage and
mononuclear cell infiltration in the late stage (39). In previous in vitro studies, we have shown inhibition of C. pneumoniae growth in HEp-2 cells pretreated with gamma interferon
and tumor necrosis factor alpha (TNF- Although isolation of C. pneumoniae from coronary and
carotid atheromas strengthens the role of this organism in
atherosclerosis, a cause-and-effect relation between C. pneumoniae and the development of atherosclerotic lesions has not
been established. Previous molecular characterization of a carotid
isolate has been performed by using Southern hybridization analysis of
genomic digests and sequence analysis of the major outer membrane
protein (MOMP) gene (omp1) (17). Similar studies
have not been documented with the coronary isolate, designated C. pneumoniae A-03.
Prior evidence has shown conservation in the omp1 genes of
several C. pneumoniae strains (9, 18, 25), while
considerable sequence variation is present among various C. trachomatis serovars. Unlike C. pneumoniae, specific
antigenic determinants that distinguish different species, subspecies,
and serovars of C. trachomatis are located in the MOMP
(1, 40). Different serovars of C. pneumoniae have
not yet been identified; however, antigenic variation among strains has
been observed by immunoblot studies (3, 35), and
serovar-specific antigenic determinants may reside in a 65-kDa protein,
as reported by Jantos et al. (18).
The present study was aimed at characterizing C. pneumoniae
A-03 at the molecular level and to examine its biological activity in
vitro. Our first goal was to determine whether diversity existed in the
omp1 gene of this strain compared to prototype strains of
C. pneumoniae. Subsequent studies focused on the ability of A-03 and three respiratory strains of C. pneumoniae to
stimulate the production of MCP-1, IL-8, and soluble ICAM-1 (sICAM-1)
in human endothelial cells in vitro.
Cell lines.
Human umbilical vein endothelial cells (HUVEC;
ATCC 1730-CRL) were maintained in Ham's F12K medium (Sigma, St. Louis,
Mo.) supplemented with 10% fetal bovine serum, 1%
penicillin-streptomycin-amphotericin B (Fungizone) mix (BioWhittaker,
Walkersville, Md.), 30 µg of endothelial cell growth supplement per
ml, and 100 µg of heparin (Sigma) per ml. Cells were grown in
75-cm2 culture flasks and transferred into 24-well plates
containing gelatin-coated glass coverslips. HUVEC were seeded at 2 × 105 cells/well and allowed to adhere overnight in media
without endothelial cell growth supplement prior to infection.
C. pneumoniae strains.
C. pneumoniae A-03
(ATCC VR-1452) was previously isolated in our laboratory from an
atheroma of a patient with coronary artery disease (30).
C. pneumoniae TW-183 and AR-39 were obtained from the
Washington Research Foundation, Seattle. C. pneumoniae
BAL-16 and T-2634 were kindly provided by Margaret Hammerschlag, State University of New York, Brooklyn. Two additional clinical isolates from
the University of Louisville, UL-029 and UL-083, were also selected for
comparative sequence analysis of omp1. Bacterial strains
were propagated in HEp-2 cell monolayers by a modification of the
procedures described by Wong et al. (38). Briefly, confluent monolayers of HEp-2 cells in 1-dram vials were infected separately with
each strain. C. pneumoniae were previously suspended in
inoculation medium (supplemented Iscove's minimal essential medium
plus 4 mg of glucose per ml [pH 7.5]) before addition to the HEp-2
monolayers. Infection was done by centrifugation at 800 × g for 1 h at 4°C. Infected cultures were then
incubated at 37°C in 5% CO2 for 30 min. The medium was
replaced with growth medium (inoculation medium plus 1 µg of
cycloheximide per ml), and infected cultures were incubated for 48 to
72 h. Sequential passages in 1-dram vials were done before
inoculation into 75-cm2 HEp-2 monolayers. C. pneumoniae was harvested by disruption of the monolayers with
sterile glass beads, sonication, and low-speed centrifugation at
200 × g. Aliquots of C. pneumoniae were
titrated and stored at omp1 gene amplification and sequencing.
DNA from
C. pneumoniae A-03, TW-183, AR-39, BAL-16, T-2634, UL-029,
and UL-083 was extracted from elementary body suspensions by lysis with
10 µg of proteinase K per ml in TE buffer (10 mM Tris-HCl [pH 8.3],
1 mM EDTA, 0.45% [vol/vol] Tween 20-Nonidet P-40) incubation at
55°C, phenol-chloroform extraction, precipitation with 95% ethanol,
and resuspension in TE as previously described (7). One
microliter of DNA was amplified by using primers that flank the
omp1 gene of C. pneumoniae (CS-F and CF-B)
(7). Automated sequencing of the amplified product was
performed with a model 377 automated sequencer (Perkin Elmer-ABI,
Foster City, Calif.) as instructed by the manufacturer.
Infection of HUVEC.
HUVEC monolayers in 24-well plates were
infected separately with C. pneumoniae A-03, TW-183, BAL-16,
and T-2634 suspended in inoculation medium. Cells were inoculated with
2 × 105 inclusion-forming units (IFU) of each strain
per well, resulting in a multiplicity of infection (MOI) of 1:1. In
other experiments, a higher inoculum concentration of 2 × 106 IFU/well (MOI of 10:1) was used for strains A-03 and
BAL-16 to study a dose-dependent activation of HUVEC. In addition to
infection with viable C. pneumoniae, HUVEC were infected
with organisms that had been previously inactivated by heat (90°C for
30 min) or UV light (12-h exposure to a model UVSL-25 Mineralight UV
lamp at a distance of 2 cm). When such preparations were examined for viable organisms in HEp-2 cell cultures, none were detected.
Inoculation of HUVEC with viable or inactivated C. pneumoniae was followed by centrifugation at 800 × g for 1 h at 4°C. After 30 min of incubation at
37°C with 5% CO2, cell monolayers were washed with
Hanks' balanced salt solution and the medium was replaced with growth
medium lacking cycloheximide. Since the bacterial inoculum may contain
remnants of HEp-2 cells, mock-infected controls were included. These
consisted of HUVEC treated with crude lysates of HEp-2 cells and were
processed in the same manner as infected cells. Uninfected,
mock-infected, and infected cells were incubated at 37°C in 5%
CO2, and culture supernatants were collected after 6, 24, and 48 h. Supernatants were stored at
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Characterization of a Strain of Chlamydia
pneumoniae Isolated from a Coronary Atheroma by Analysis of the
omp1 Gene and Biological Activity in Human
Endothelial Cells
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
) (34). Induction of
proinflammatory cytokines such as TNF-, IL-1, and IL-6 by C. pneumoniae has been studied in human monocytic cells
(16). The capability of C. pneumoniae to
replicate in vitro in human endothelial cells, aortic smooth muscle
cells, and macrophages has been shown previously (8, 10, 11,
19). Infection of endothelial cells results in the stimulation of
adhesion molecules such as endothelial-leukocyte adhesion molecule 1, ICAM-1, and vascular cell adhesion molecule 1 (20). Data
concerning the production of chemokines such as IL-8 and MCP-1 by
endothelial cells in response to C. pneumoniae infection
have not been reported.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
70°C in inoculation medium.
20°C until chemokine and
sICAM-1 measurements were performed. The response of HUVEC to
stimulation with 500 U of human recombinant TNF- (Promega, Madison,
Wis.) per ml was used as a positive control.
Chemokine and adhesion molecule measurements.
Levels of
MCP-1, IL-8, and sICAM-1 were measured from the supernatants of
uninfected, mock-infected, infected, and TNF--treated cells by
commercially available enzyme-linked immunosorbent assay (ELISA) kits
(R&D Systems, Minneapolis, Minn.). The ELISAs were performed according
to the manufacturer's instructions.
Data analysis. Raw data of experimental groups from the ELISAs were subjected to analysis of variance with the Tukey-HSD multiple-comparison test. A P of <0.05 was used as the alpha value to determine statistical significance for all analyses.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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omp1 sequence analysis of C. pneumoniae strains. The sequence of the omp1 gene of the coronary atheroma C. pneumoniae A-03 strain was determined and compared to the gene sequences of six respiratory strains of C. pneumoniae. The omp1 sequences of C. pneumoniae BAL-16, T-2634, and two clinical isolates from the University of Louisville (UL-029 and UL-083) were identical to those of the prototype strains TW-183 and AR-39. Figure 1 shows comparison of the omp1 gene sequences of C. pneumoniae A-03 and BAL-16. Six nucleotide changes were noted in the omp1 gene sequence of C. pneumoniae A-03, with five of these resulting in nonsynonymous codons. One substitution was found in each of the variable segments 1, 2, and 3 (VS1, -2, and -3), while the remaining two localized at conserved regions of omp1. VS4 sequences were identical among all strains examined. Significantly, the amino acid change in VS1 of C. pneumoniae A-03 (Ser to Pro at position 61) was nonconservative. The remaining amino acid changes were synonymous.
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Secretion of MCP-1 and IL-8 by HUVEC in response to C. pneumoniae infection.
The in vitro activities of C. pneumoniae A-03 and the respiratory strains BAL-16, T-2634, and
TW-183 were examined in infected endothelial cells by analysis of the
production of the chemokines MCP-1 and IL-8. The kinetics of MCP-1 and
IL-8 secretion were determined from HUVEC infected with C. pneumoniae A-03 and BAL-16 at MOIs of 1:1 and 10:1 after 6, 24, and 48 h of incubation. As shown in Fig.
2A, secretion of MCP-1 was induced in a
time-dependent fashion in response to C. pneumoniae A-03 and
BAL-16. At 6 h p.i., levels of MCP-1 were not higher than those in
mock-infected controls, even at an MOI of 10:1. Following 24 and
48 h of infection, significant increases in MCP-1 production were
observed in response to C. pneumoniae BAL-16 at both MOIs
compared to mock-infected controls. These increases were approximately
fivefold at 24 h (P < 0.01) and threefold at
48 h (P < 0.002). In contrast, induction of MCP-1 by C. pneumoniae A-03 did not reach statistical significance
compared to mock-infected controls, even at an MOI of 10:1. After
24 h of infection, levels of MCP-1 increased approximately
threefold in response to A-03 at both MOIs and remained elevated after
48 h only with an MOI of 10:1. Concentrations of MCP-1 in
uninfected cells ranged from 0.2 to 0.5 ng/ml, while treatment of cells
with 500 U of human recombinant TNF- per ml, used as a positive
control, increased MCP-1 production from 0.8 to 12.5 ng/ml during the
48-h length of the assays.
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-treated cells secreted 1.1 to 18.2 ng of IL-8 per ml during the experiments.
In addition to C. pneumoniae A-03 and BAL-16, strains TW-183
and T-2634 were examined for the ability to induce MCP-1 and IL-8
production in HUVEC. As seen in Fig. 3,
the extent of MCP-1 and IL-8 secretion by HUVEC infected at an MOI of
1:1 depended on the C. pneumoniae strain examined. There was
a significant difference in the relative amounts of MCP-1 and IL-8
stimulated by strains TW-183 and T-2634 as measured at 24 h p.i.
compared to strains A-03 and BAL-16 (P < 0.001 in Fig.
3A and B, respectively). Differences in the induction of these proteins
between strains displaying the lowest and highest stimulatory
activities (A-03 and T-2634) were approximately 11-fold for MCP-1 and
6-fold for IL-8.
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Production of sICAM-1 by HUVEC in response to C. pneumoniae infection.
The in vitro activities of C. pneumoniae A-03, BAL-16, TW-183, and T-2634 were also examined by
analysis of sICAM-1 production by infected HUVEC. Figure
4 depicts the kinetics of sICAM-1
production in response to infection with C. pneumoniae A-03
and BAL-16. At 24 h p.i., strain BAL-16 stimulated significant
increases in sICAM-1 production compared to mock-infected controls at
both MOIs. These increases were approximately fivefold at an MOI of 1:1
(P < 0.03) and threefold at an MOI of 10:1
(P < 0.004). Contrary to this, infection with C. pneumoniae A-03 caused little if any increase in sICAM-1
production after 24 and 48 h, even at an MOI of 10:1. Elevated
levels of sICAM-1 were maintained at 48 h p.i. in cells infected
with strain BAL-16 at both MOIs (P < 0.02 for an MOI of 10:1). In comparison to the MCP-1 experiments, raising the MOI from
1:1 to 10:1 did not increase levels of sICAM-1 stimulation by both
strains at 24 h. Concentrations of sICAM-1 from uninfected HUVEC
were comparable to those for mock-infected controls, whereas production
of this protein from cells treated with 500 U of TNF- per ml
fluctuated from 2.1 to 22 ng/ml throughout the experiments.
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Growth of C. pneumoniae in HUVEC. HUVEC supported similar replication of all four strains of C. pneumoniae examined, with growth titers ranging from 6.5 × 103 to 9.8 × 103 IFU/ml when an MOI of 1:1 was used. At the same MOI, numbers of chlamydial inclusion bodies per HPF, as well as the percentages of HUVEC with one or more inclusion body per HPF, were similar among all strains after 48 h of infection (data not shown). Inactivation of C. pneumoniae by heat or UV light resulted in nonproductive infection. All C. pneumoniae strains had similar effects on viability of infected HUVEC at 24 (>80%) or 48 h p.i. (>70%), as determined by trypan blue dye exclusion analysis.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Infections caused by C. pneumoniae are generally localized to the respiratory tract, are frequently asymptomatic, and may become chronic following acute illness despite appropriate antibiotic therapy (15). A role for C. pneumoniae in chronic inflammatory diseases has been suggested by data showing a propensity for patients with previous respiratory infection to develop asthmatic bronchitis (14) and by the physical and serological evidence implicating an association of this organism with atherosclerosis (21, 22, 24, 26, 29, 32, 33).
Isolation of C. pneumoniae from human carotid (17) and coronary (30) atheromas provides further support for an association of this bacterium with atherosclerosis by demonstrating the presence of viable organisms within lesions. Previous analysis of the MOMP gene sequence (omp1) of a carotid isolate indicated no differences with prototype C. pneumoniae respiratory strains (17). The present study demonstrates that C. pneumoniae coronary atheroma strain A-03 displays omp1 divergence from prototype respiratory strains, as well as from four additional respiratory isolates. In agreement with previous reports showing conservation of omp1 sequences among different respiratory strains of C. pneumoniae (9, 18, 25), we found the omp1 gene sequences of respiratory strains BAL-16, T-2634, UL-029, and UL-083 to be identical to those of the prototype strains TW-183 and AR-39. Among the nucleotide changes observed in the omp1 gene sequence of C. pneumoniae A-03, three were located in the variable segments with one resulting in a nonconservative amino acid substitution in the VS1 region.
For C. trachomatis serovars, the diversity of nucleotide sequences within the variable segments of omp1 genes accounts for the antigenic variation of the MOMP (37). Epitope mapping of the C. trachomatis MOMP has shown that three of the four variable segments (VS1, VS2, and VS4) contain serovar-, subspecies-, and species-specific antigenic determinants (1, 40). In contrast, the genetic homogeneity between different respiratory strains of C. pneumoniae in the omp1 gene may correlate with the inability to identify specific antigenic determinants in the MOMP. Previous immunoblot analyses of C. pneumoniae respiratory strains with anti-C. pneumoniae rabbit sera or human sera from patients with C. pneumoniae infection have shown that the recognition of the MOMP is genus reactive (5, 6). Even though evidence suggests that the C. pneumoniae MOMP is not immunodominant (6), further characterization of C. pneumoniae A-03 by serological studies is required to determine whether the omp1 substitutions correlate with distinct antigenic reactivities in the MOMP of this strain.
The results from our in vitro studies demonstrate that infection of endothelial cells with different strains of C. pneumoniae elicits the production of MCP-1, IL-8, and sICAM. Induction of these proteins in HUVEC was time dependent, with no increases detected early after infection (6 h) in response to C. pneumoniae A-03 and BAL-16. The extent of endothelial cell stimulation by C. pneumoniae A-03, BAL-16, TW-183, and T-2634 was strain dependent at low MOIs following 24 h of incubation. Strain A-03 exhibited the lowest stimulatory activity, while T-2634 induced the highest levels of MCP-1, IL-8, and sICAM-1 among all strains examined. The heterogeneity observed among these strains in the activation of HUVEC could not be explained by differences in the extent of chlamydial replication, since similar growth titers, as well as numbers of inclusion-containing cells, were observed for all four strains. These findings may reflect intrinsic differences among strains of C. pneumoniae in the activation of endothelial cells in vitro.
A variety of mechanisms may be involved in the stimulation of
chemokines and adhesion molecules by C. pneumoniae in
endothelial cells. Activation of HUVEC by C. pneumoniae may
involve an autocrine pathway via IL-1 or TNF- production in a
fashion similar to that described for C. trachomatis and
C. psittaci in epithelial cells (31). The results
obtained with heat-inactivated and UV-inactivated organisms suggest
that a heat-labile component may be required for the activation of
HUVEC by C. pneumoniae. Analogous to what has been described
for C. trachomatis and C. psittaci entry of HeLa
cells and L cells (4), UV-inactivated C. pneumoniae may still possess the ability to invade endothelial
cells, thereby activating a cascade of signaling mechanisms associated
with phagocytosis. The complete inhibition of MCP-1, IL-8, and sICAM-1
stimulation by heat treatment of C. pneumoniae suggests that
chlamydial lipopolysaccharide may not be important in eliciting the
production of these inflammatory mediators from endothelial cells.
The increased production of chemokines and adhesion molecules by endothelial cells in response to C. pneumoniae infection may be important for recruiting leukocytes to the site of infection. This event presumably would result in both protective and deleterious consequences since the inflammatory response can lead to clearance of the infection as well as contribute to tissue damage. Although a casual relationship between C. pneumoniae and the development of atherosclerosis awaits further investigations, the ability of this organism to trigger an inflammatory response from endothelial cells suggests a potential role for C. pneumoniae in the pathology associated with this disease.
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ACKNOWLEDGMENTS |
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This study was supported by a grant from the Heart and Lung Institute, Jewish Hospital Foundation, Louisville, Ky.
We thank Linda Jane Goldsmith, University of Louisville Biostatistics Center, for assistance in statistical analysis of data.
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FOOTNOTES |
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* Corresponding author. Mailing address: Division of Infectious Diseases, MDR Building, Room 622, Department of Medicine, University of Louisville, Louisville, KY 40292. Phone: (502) 852-5132. Fax: (502) 852-1147. E-mail: JTSUMM01{at}ulkyvm.louisville.edu.
Editor: J. G. Cannon
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Discussion
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Infect Immun, April 1998, p. 1370-1376, Vol. 66, No. 4
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