© 2001 American Heart Association, Inc.
Vascular Biology |
Chlamydia pneumoniae Proteins Induce Secretion of the 92-kDa Gelatinase by Human Monocyte– Derived Macrophages
From the Wihuri Research Institute (P.V.-K., P.T.K.); the Haartman Institute (M.P.), Department of Virology, University of Helsinki; and the National Public Health Institute (M.S.), Department of Vaccines, Helsinki, Finland; and Parke-Davis Pharmaceuticals Research (H.G.W.), Ann Arbor, Mich.
Correspondence to Petri T. Kovanen, MD, PhD, Wihuri Research Institute, Kalliolinnantie 4, 00140 Helsinki, Finland. E-mail Petri.Kovanen{at}wri.fi
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract—Chlamydia pneumoniae, an intracellular Gram-negative respiratory bacterium, and macrophages are present in inflammatory tissue sites such as atherosclerotic lesions, where abnormal degradation of the extracellular matrix takes place. To evaluate the potential of C pneumoniae for participation in matrix destruction, we studied the effect of this bacterium on the production of 3 matrix-degrading metalloproteinases, 92-kDa gelatinase, interstitial collagenase-1, and stromelysin-1, and their natural inhibitor TIMP-1 (tissue inhibitor of metalloproteinases-1) by human monocyte–derived macrophages differentiated in vitro. Spontaneous production of collagenase and stromelysin by these cells was minimal and was not influenced by C pneumoniae. In contrast, the cells secreted substantial basal quantities of 92-kDa gelatinase, the secretion of which was stimulated (on average, 2.5-fold) by C pneumoniae. C pneumoniae regulated the expression of 92-kDa gelatinase by macrophages at the pretranslational level. Macrophages secreted only small quantities of TIMP-1. The chlamydial proteins Omp2, MOMP, and HSP60 were also found to participate in the induction of 92-kDa gelatinase by C pneumoniae. Denaturation of chlamydial proteins by boiling reduced 92-kDa gelatinase secretion only partially (by 35%), suggesting that the heat-stabile lipopolysaccharide molecules also stimulate secretion of the enzyme. The results show that production of 92-kDa gelatinase by human macrophages is selectively upregulated by C pneumoniae, which suggests that these bacteria, when present in a macrophage-containing inflammatory environment, actively participate in the destruction of the extracellular matrix.
Key Words: humans • monocytes • macrophages • cellular activation • inflammatory mediators
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Matrix metalloproteinases (MMPs) are structurally related and participate in the degradation of extracellular matrix components.1 At present, the MMP gene family consists of at least 17 zinc-dependent endopeptidases, which can be divided into subgroups of collagenases, gelatinases, stromelysins, and membrane-type MMPs.1 Besides participating in normal homeodynamics and developmental remodeling of connective tissues, the MMPs appear to contribute significantly, by their proteolytic activity, to the tissue damage seen in chronic inflammatory diseases such as rheumatoid arthritis,2 osteoarthritis,3 and atherosclerosis.4 MMPs, in most instances, are not produced constitutively, but their expression is induced in a cell type–specific manner by inflammatory mediators such as cytokines,5 cellular transformation,6 hormones,7 growth factors,8 and bacterial lipopolysaccharide (LPS).9
The MMPs secreted by macrophages include interstitial collagenase (MMP-1),10 72-kDa gelatinase (MMP-2),6 stromelysin-1 (MMP-3),8 matrilysin (MMP-7),11 92-kDa gelatinase (MMP-9),12 and a unique elastase designated macrophage metalloelastase (MMP-12).13 Macrophages also produce 2 specific inhibitors of MMPs, known as tissue inhibitors of metalloproteinases-1 and -2 (TIMP-1 and TIMP-2).9 14 Of these, TIMP-1 interacts with the active forms of interstitial collagenase and stromelysin as well as with the active and latent forms of 92-kDa gelatinase.15
C pneumoniae is an obligate, intracellular, Gram-negative
bacterium that is distinguished from other bacteria by a unique growth
cycle.16 In this growth cycle there are 2
morphologically and functionally distinct cell types: the infectious
elementary body (EB) and the reproductive reticulate body
(RB).16 The outer envelope of the elementary body is
composed of cysteine-rich structural proteins with molecular masses of
98, 60-doublet, 39.5, and 15.5 kDa.17 The 39.5-kDa major
outer membrane protein (MOMP) is considered the primary target of
immune response in C trachomatis, but in C
pneumoniae infection, MOMP is less
immunogenic.18 Outer membrane protein 2 (Omp2) is a
second constituent of the chlamydial outer membrane complex; it has a
molecular mass of 
60 kDa.19 Omp2 has been found to be a
major immunogen of C pneumoniae.19 Yet another
60-kDa protein has been identified in C pneumoniae; it is a
homolog of C trachomatis GroEL and a member of the heat
shock protein family, HSP60.20 C pneumoniae are
able to multiply within macrophages, where they persist for
long periods without causing any damage until they are
reactivated by immunosuppression16 or by
coincidental infection with other organisms.
C pneumoniae has been implicated as a causative agent of
several common respiratory infections, especially
pneumonias.16 Importantly, a serological association
was also found between C pneumoniae infection and
coronary heart disease.21 Interestingly,
C pneumoniae has been identified within macrophages
and smooth muscle cells of atherosclerotic coronary artery
specimens at necropsy.22 However, the mechanisms by
which C pneumoniae affects the natural history of
atherogenesis remain poorly understood. Very recently, Kol et
al23 reported the presence of HSP60 of C
pneumoniae in human carotid atheromas. These
investigators also reported that chlamydial HSP60 derived from C
trachomatis stimulates the expression of tumor necrosis factor-
(TNF-) and MMP-9 by mouse peritoneal
macrophages.23
In our study, we have examined the capacity of live C pneumoniae bacteria and some of its protein components to influence the production of interstitial collagenase, stromelysin, 92-kDa gelatinase, and TIMP-1 by human monocyte–derived macrophages. C pneumoniae induced 92-kDa gelatinase expression by macrophages but had no influence on the production of interstitial collagenase or stromelysin. Stimulation of monocyte-derived macrophage 92-kDa gelatinase production by C pneumoniae occurred via the chlamydial-derived proteins Omp2, MOMP, and HSP60.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Reagents
Ficoll-Paque was purchased from Pharmacia LKB Biotechnology. Macrophage serum-free medium, RPMI 1640, Dulbecco’s modified Eagle’s medium, Dulbecco’s PBS without Ca2+ and Mg2+, fetal bovine serum, and penicillin/streptomycin (10 000 IU/mL and 10 000 µg/mL, respectively) were obtained from GIBCO. Human recombinant granulocyte macrophage colony-stimulating factor (GM-CSF) was generously provided by Novartis Oy (Espoo, Finland). LPS, derived from Escherichia coli serotype 0111:B4, ethidium bromide, Coomassie brilliant blue R-250, calf thymus DNA, and Triton X-100 were purchased from the Sigma Chemical Co. Gelatin was obtained from Merck KGaA. Meglumine diatrizoate (Urografin) was from Schering AG. C trachomatis C and L2 were purchased from Biological Materials Distribution Center, Washington Research Foundation. All other chemicals used were of reagent grade.
Isolation and Culture of Macrophages
Human monocytes from healthy control subjects were isolated from
buffy coat cells (obtained from the Finnish Red Cross Blood Transfusion
Service, Helsinki, Finland) by Ficoll-Paque as described
earlier.5 The culture medium was replaced with fresh
serum-free medium, and supplemented with 100 U/mL penicillin, 100
µg/mL streptomycin, and 10 ng/mL GM-CSF (medium A) after 24 hours and
every 48 hours thereafter. Experiments were started either with freshly
isolated monocytes (day 0 of culture) or with monocytes that had been
cultured for 7 days in vitro. These monocytes that had differentiated
in vitro are hereafter referred to macrophages.
Growth and Purification of C pneumoniae
A Finnish Mycoplasma-free (no Mycoplasma
DNA detected by polymerase chain reaction [PCR]) C
pneumoniae isolate K-6 was propagated in HL cells. Infected cells
were collected and sonicated, and the bacteria were purified through a
gradient of meglumine diatrizoate.24 Purified
organisms were resuspended in sucrose/phosphate/glutamic acid and
stored in aliquots at -70°C until used. The infectivity, measured as
inclusion-forming units (IFUs) of the bacterial preparation, was
determined in cycloheximide-treated HL cells.25
Recombinant C pneumoniae Proteins
C pneumoniae proteins MOMP, Omp2, and HSP60 were
produced in the heterologous host, Bacillus subtilis, strain
168, which is apathogenic and secretes no toxins. As a Gram-positive
bacterium it is devoid of endotoxin (LPS). Protein production and
purification have been described in detail by U. Airaksinen et al
(unpublished data, 1998). Briefly, genes encoding the above proteins
were amplified by PCR and inserted into an expression vector for
intracellular production. HSP60 was purified with affinity
chromatography based on His-tag in native state. Both MOMP and Omp2
were found in B subtilis as inclusion bodies, and they were
purified after denaturation with urea; there was no renaturation
step.
Treatment of Macrophages With C pneumoniae
and Its Proteins
Before experiments, each well of macrophages was washed
3 times with PBS, and 1 mL of fresh medium A was added. The cells were
then incubated for 48 hours in either the absence or presence of
C pneumoniae. In a third series of wells, the
macrophages were exposed to the indicated doses of C
pneumoniae for either 48 or 1.5 hours, after which the medium was
replaced by fresh medium that did not contain C pneumoniae.
In other experiments, macrophages were incubated with the
indicated doses of chlamydial proteins for 48 hours. After incubation,
the conditioned media were collected, centrifuged at
400g for 5 minutes at 20°C, and kept frozen at -20°C
until their MMP or TIMP contents were quantified by ELISA. For
zymography, the macrophages were incubated for 48 hours in a
protein-free medium consisting of RPMI supplemented with 10 ng/mL
GM-CSF, 100 U/mL penicillin, and 100 µg/mL streptomycin (medium
B).
Gelatin Zymography
Gelatinolytic activity was assessed by
SDS–polyacrylamide gel electrophoresis of conditioned medium
(10 µL) under nonreducing conditions in gels containing 10%
polyacrylamide and 1 mg/mL gelatin. To renature the proteins
after electrophoresis, SDS was removed from the gels by washing them
twice for 15 minutes at room temperature in 2.5% Triton X-100. Gels
were then washed once with buffer containing 50 mmol/L Tris (pH
8.2), 5 mmol/L CaCl2, and 0.5 µmol/L
ZnCl2, after which the gels were incubated in
this buffer overnight at 37°C. To detect bands with
gelatinolytic activity, gels were stained with
0.5% Coomassie brilliant blue R-250. For molecular-weight
standardization, we used a low-molecular-weight standard from
Pharmacia.
Immunological Assays
Competitive-binding ELISAs for interstitial
collagenase, stromelysin, 92-kDa gelatinase, and TIMP-1
were performed on cell supernatants, as described
previously.5 These assays are sensitive in the nanogram
range and use polyclonal antibodies that recognize the free enzymes
with avidities equal to those of the enzymes complexed with their
substrates or with the TIMPs.
Reverse Transcription (RT)–PCR Analysis of 92-kDa
Gelatinase Expression
Total RNA was isolated from macrophages by using an
ultra pure TRIzol reagent (GIBCO). The RNA was transcribed into cDNA
with a Superscript preamplification system (GIBCO BRL). The obtained
cDNA was further amplified by PCR with specific
oligonucleotides for human 92-kDa gelatinase (sense,
5'-CACTGTCCACCC-CTCAGAGC-3'; antisense
5'-GCCACTTGTCGGCGATAAGG-3').27 In a control experiment,
the expression level of a housekeeping gene,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH; sense,
5'-ACCACAGTCCATGCCATCAC-3'; antisense, 5'-TCCACCAC-CCTGTTGCTGTA-3',
generating a 452-bp DNA fragment, was found to be comparable both in
the control and stimulated macrophages. PCR was performed under
the following conditions: for 92-kDa gelatinase, 94°C (1 minute) for
1 cycle; 94°C (30 seconds), 63°C (1 minute), and 72°C (45
seconds) for 40 cycles; and the final incubation at 72°C for 10
minutes; for GAPDH, 94°C (1 minute) for 1 cycle; 94°C (30 seconds),
65°C (30 seconds), and 72°C (30 seconds) for 30 cycles; and the
final incubation at 72°C for 10 minutes. Aliquots (7 µL) of the PCR
product were electrophoresed through 1.2% agarose gels containing
0.4 µg/mL ethidium bromide. Gels were illuminated with UV light and
photographed with Polaroid film (Polaroid Ltd, UK).
Measurement of Cellular DNA
After incubations, the cells in each well were washed and
dissolved in 0.5 mL of 0.2% Triton X-100, 1 mmol/L NaOH, for
assay of their DNA content.28 Calf thymus DNA was used as
a standard.
Statistical Analysis
Results were analyzed by using a random-effects model
for repeated measurements.29 These models take into
account that repeated measurement from the same individual are
correlated. For all models, the random intercept for each individual
was fitted to adjust for between-individual variability.29
Differences were considered to be statistically significant at a
P value <0.05.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Effect of C pneumoniae Bacterium on Production of 92-kDa Gelatinase, Interstitial Collagenase, Stromelysin-1, and TIMP-1
Blood monocytes obtained from healthy individuals were allowed to differentiate into macrophages, and then the effect of C pneumoniae on their ability to secrete 3 different MMPs was studied: 92-kDa gelatinase (MMP-9), interstitial collagenase (MMP-1), and stromelysin (MMP-3) (Figure 1
). The macrophages spontaneously
released substantial amounts of 92-kDa gelatinase (first panel), a
finding consistent with a previous report.6
Among these donors, some variability was noted in the basal
production of the enzyme (0.14 to 0.35 µg 92-kDa gelatinase
per µg cellular DNA/48 hours). C pneumoniae induced
dose-dependent stimulation of 92-kDa gelatinase secretion. Notably, the
cells from each donor (n=5) were stimulated by C pneumoniae,
the stimulatory effect varying from 1.3- to 3.6-fold. At the largest
amount of C pneumoniae used
(3x104 IFU), secretion of the enzyme was
augmented, on average, 2.5-fold. For comparison, the effect of E
coli LPS, an established stimulator of macrophage MMP
production, was also studied. The same degree of induction
(2.5-fold) was also seen with E coli LPS. In contrast to
the 92-kDa gelatinase secretion, C pneumoniae bacteria had
no significant effect on the basal secretion of
interstitial collagenase (second panel),
stromelysin (third panel), or TIMP-1 (fourth panel).
|
Zymographic Analysis of 92-kDa Gelatinase From
Macrophages Stimulated With C
pneumoniae
Gelatinase (92-kDa) was present on the zymograms as a single
band migrating at 90 kDa (Figure 2).
C pneumoniae induced 92-kDa gelatinase production in
a concentration-dependent fashion within the range of IFUs studied
(3x102 to 3x106 IFU).
|
Pretranslational Control of 92-kDa Gelatinase Expression by
C pneumoniae
To study the mechanisms of the action of C pneumoniae
on macrophages, mRNA levels of 92-kDa gelatinase were examined
by PCR. Gelatinase (92-kDa) was detected as the expected 243-bp
product.27 The RT-PCR signal for 92-kDa
gelatinase was significantly increased after C pneumoniae
stimulation (Figure 3).
|
Effects of Different Exposure Times to C
pneumoniae on 92-kDa Gelatinase Secretion
In the above experiments, the human macrophages had been
exposed to C pneumoniae for 48 hours. We also tested whether
shorter contacts of the macrophages with C
pneumoniae would be sufficient to induce 92-kDa gelatinase
secretion. In this experiment, either the macrophages were
incubated for 1.5 hours, after which the culture medium was changed and
incubation continued for a total length of 48 hours, or C
pneumoniae was allowed to stimulate the macrophages for
the entire length of the incubation (48 hours) (Figure 4). When the bacteria were incubated
with the macrophages for 1.5 hours, the stimulatory effect on
92-kDa gelatinase secretion by the cells was almost the same as when
the bacteria were incubated with the cells for 48 hours.
|
Effects of Chlamydial Proteins Omp2, MOMP, and HSP60 on 92-kDa
Gelatinase Secretion
Macrophages were incubated with the indicated doses of the
chlamydial proteins Omp2, MOMP, and HSP60 for 48 hours (Figure 5). Omp2 increased a dose-dependent
stimulation of 92-kDa gelatinase production, maximally
5-fold. Chlamydial MOMP also induced 92-kDa gelatinase secretion by
the cells, but in sharp contrast to Omp2, the maximal effect was found
at the lowest concentration used (0.1 µg/mL), which increased 92-kDa
gelatinase by 5-fold. When higher concentrations were used (up to 2
µg/mL), a dose-dependent inhibition of stimulated 92-kDa gelatinase
secretion was observed back to basal levels. If higher concentrations
were used, MOMP was cytotoxic to the cells. HSP60 of C
pneumoniae induced 92-kDa gelatinase production, the
near-maximal effect obtained at the lowest concentration used (0.1
µg/mL, Figure 5). Induction of 92-kDa gelatinase secretion by
HSP60 was maximally 2- to 3-fold, which equaled that produced by
E coli LPS (data not shown). No induction of 92-kDa
gelatinase was noted when the macrophages were incubated with
the corresponding buffers without proteins.
|
P<0.002 concentration used as continuous variable.
Effects of Different Exposure Times to MOMP, Omp2, and HSP60 on
92-kDa Gelatinase Secretion
The above experiment with the 3 chlamydial proteins Omp2, MOMP,
and HSP60 was then repeated with freshly prepared monocyte-derived
macrophages derived from 3 different subjects (Figure 6). In this experiment, the
macrophages were exposed to 3 different concentrations of the
proteins for either 1.5 or 48 hours and using the same protocol as
described for the experiment shown in Figure 4
. Comparison of
panels A and B in Figure 5 reveals similar patterns of
upregulation of the 92-kDa gelatinase after short and prolonged
antigenic stimulation of the macrophages. Moreover, the results
were similar to those seen in Figure 5, with MOMP possessing the
strongest stimulatory effect at the lowest and Omp2 at the highest
concentration used.
|
Effect of Heat Treatment on C
pneumoniae– Induced 92-kDa Gelatinase Secretion
To further evaluate which C pneumoniae components
participated in the induction of 92-kDa gelatinase by
macrophages, the bacteria were boiled for 20 minutes before
addition to the cell cultures (Table). Heat
treatment of C pneumoniae reduced the effect on 92-kDa
gelatinase secretion by these bacteria moderately, on average, by 35%
(from a value of 0.42 to a value of 0.27 µg/µg cellular DNA per 48
hours, considering negative control as the background activity to be
subtracted). This result was observed in each of the studied (n=3)
buffy coats and strongly suggests that chlamydial LPS, which is
thermostable (in contrast to the proteins), also participates in the
induction of 92-kDa gelatinase production.
|
Effect of Monocyte Differentiation on Basal and C
pneumoniae–Induced 92-kDa Gelatinase Secretion
Production of 92-kDa gelatinase was studied as a function
of cellular differentiation by comparing freshly harvested human
monocytes with cells from th same source differentiated in vitro for 7
days. As shown in Figure 7A, the
unstimulated control monocytes secreted detectable quantities of 92-kDa
gelatinase, and this basal production was increased (by
6-fold) when the cells were allowed to differentiate into
macrophages (Figure 7B). In both monocytes and
monocyte-derived macrophages, constitutive secretion was
stimulated by C pneumoniae (on average 2.5-fold). The same
degree of stimulation was seen with E coli LPS. These
results show that secretion of 92-kDa gelatinase by blood monocytes
increases with cellular differentiation and that substantially larger
amounts of the enzyme are secreted by C pneumoniae–treated
macrophages than by C pneumoniae–treated
monocytes.
|
Comparison of the Effects of C trachomatis Serotypes
C and L2 and of C pneumoniae on 92-kDa
Gelatinase Production
The effects of C trachomatis serotypes C and
L2 and of C pneumoniae on 92-kDa
gelatinase production by human monocyte–derived
macrophages are shown in Figure 8. Interestingly, C
trachomatis serotypes C and L2 also
increased 92-kDa gelatinase secretion by human monocyte–derived
macrophages. C trachomatis L2
induced 92-kDa gelatinase secretion by macrophages in a
dose-dependent manner, with maximal stimulation being, on average,
4.7-fold. C trachomatis C also stimulated 92-kDa
gelatinase production but had its maximal stimulatory effect
with smaller amounts of bacteria, and secretion of the enzyme was
augmented, on average, by only 2.7-fold. In this experiment, the
stimulatory effect of E coli LPS on 92-kDa gelatinase
secretion was 5-fold (not shown).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The data presented demonstrate that C pneumoniae can increase the capacity of macrophages to produce 92-kDa gelatinase by stimulating the expression of this enzyme. Such increase in the expression of 92-kDa gelatinase could result in increased proteolytic activity in the extracellular microenvironment of the stimulated macrophages, leading to enhanced extracellular remodeling. In present work, C pneumoniae bacteria did induce both secretion and transcription of 92-kDa gelatinase. We did not investigate the mechanism of gene activation, but some information on the conditions required for activation is available. The promoter region of the gene for 92-kDa gelatinase contains nuclear factor (NF)-
B binding sites, which play a critical role in the expression
of this gene.30 Interestingly, oxidized LDL induces
92-kDa gelatinase expression in human monocyte–derived
macrophages, and an increase in 92-kDa gelatinase expression
was associated with nuclear binding of NF-B to specific sequences in
the promoter region of the 92-kDa gelatinase gene.31
Therefore, it is possible that the C pneumoniae–mediated
effect on 92-kDa gelatinase expression was due to activation of
NF-B. This possibility is supported by a recent observation
demonstrating that C pneumoniae infection of vascular smooth
muscle and endothelial cells activates
NF-B.32 The other intriguing question relates to
the finding that C pneumoniae stimulated secretion of 92-kDa
gelatinase but not secretion of interstitial
collagenase or of stromelysin. In vitro, stimulation of all
these 3 MMPs by an extracellular mediator depends on activation and
binding of activator protein (AP)-1 to specific sequences
in their promoters.33 However, induction of the
92-kDa gelatinase gene transcription also requires activation of
NF-B.33 If C pneumoniae activated
NF-B without activating other factors essential in regulation of the
other MMPs, infection of macrophages would result in selective
induction of 92-kDa gelatinase. Further studies are required to resolve
this issue.
We found that at least 3 proteins, Omp2, MOMP, and HSP60, induced
92-kDa gelatinase secretion by macrophages, Omp2 being the
primary antigen responsible for the upregulation of 92-kDa gelatinase.
Of special interest is the chlamydial HSP60, since it has been found to
colocalize with its homolog, human HPS60, within plaque
macrophages.23 However, since denaturation of the
chlamydial proteins by boiling the bacteria reduced 92-kDa gelatinase
secretion by only 35%, the residual 65% of remaining inducing
capacity is likely to be due to the heat-stable LPS molecules that are
present in the bacteria. Our study parallels the study performed in
murine macrophages, in which HSP60 was found to upregulate
92-kDa gelatinase.23 In this respect, the responses of
human and murine macrophages are similar. However, the relative
potencies of the various antigens and the specificity of their effect
on MMP secretion remain to be studied in the mouse system.
Incubation with the bacteria for as little as 1.5 hours, which is enough to allow their ingestion,34 fully induced 92-kDa gelatinase production by the macrophages. Also, the various chlamydial proteins were able to induce secretion of 92-kDa gelatinase by macrophages even after incubation for 1.5 hours. Unfortunately, we were unable to create conditions in which phagocytosis of the bacteria had been blocked. Therefore, we do not know whether the various chlamydial proteins and LPS can induce the 92-kDa gelatinase production by the macrophages on contact of the bacteria with the cell surface or whether the bacteria have to be ingested before they can induce secretion of the enzyme. Interestingly, C trachomatis also induced 92-kDa gelatinase production by monocyte-derived macrophages, and the stimulation was strong with serotype L2, which is known to be able to infect mononuclear cells, but only modest with serotype C, which does not infect mononuclear cells.35 These observations suggest that the 92-kDa gelatinase induction in monocyte-derived macrophages is a specific response of those chlamydial types capable of being phagocytized by mononuclear cells and capable of causing infection (C pneumoniae, C trachomatis L2). Importantly, addition of latex beads did not induce 92-kDa gelatinase production, demonstrating that phagocytosis alone is not sufficient for this response. In atherosclerotic plaques, C pneumoniae normally reside inside macrophages,22 and in this respect, the in vitro conditions used were similar to those in the plaques. After exposure to the live bacteria, fewer than 1% of the macrophages contained C pneumoniae inclusions (indicative of replicating bacteria) even after incubation for 48 hours. We do not know whether this small fraction of cells was responsible for the entire secretion of 92-kDa gelatinase. However, since even killed C pneumoniae can induce the production of 92-kDa gelatinase,23 mere ingestion of C pneumoniae without replication appears to be sufficient for induction of the enzyme. Since the phagocytosed bacteria are at various developmental stages and, accordingly, some of them may not form inclusions, the fraction of macrophages that contained C pneumoniae and contributed to the production of 92-kDa gelatinase is likely to be higher than 1%. Finally, Haemophilus influenzae, which, like C pneumoniae and E coli, is also a Gram-negative LPS-containing bacterium, failed to induce production of 92-kDa gelatinase (R. Pastila et al, unpublished observations, 1977). Thus, not any infective agent may provoke 92-kDa gelatinase induction in monocyte-derived macrophages in humans.
The activity of metalloproteinases on substrates of the extracellular matrix depends on the balance existing between these enzymes and their endogenous inhibitors, the TIMPs. Since TIMP expression can be regulated by biological agents such as cytokines,15 we tested the ability of C pneumoniae to stimulate the production of TIMP-1 by human macrophages, the specific inhibitor of the MMPs investigated in this study. Although the degree of stimulation of TIMP-1 secretion was almost as high as that of 92-kDa gelatinase secretion, it was statistically not significant, and the basal secretion of TIMP-1 was so low that even the stimulated secretion was not high enough to even approach the 1:1 molar ratio required for full inhibition of 92-kDa gelatinase. Indeed, the molar expression of TIMP-1 was only about one fifth the expression of 92-kDa gelatinase, revealing that an excess of enzyme molecules was released from the stimulated macrophages.
Numerous studies have revealed the presence of C pneumoniae
in coronary
atheromas.21 22 36 37 38 Moreover,
immunostaining has shown that in both unstable and
stable angina, 92-kDa gelatinase production in arteries is
increased, whereas in normal arteries 92-kDa gelatinase is not
expressed.39 Increased production of 92-kDa
gelatinase in atherosclerosis may contribute to the
matrix destruction that leads to plaque rupture. Production of
92-kDa gelatinase by macrophages has recently been observed in
human aortic aneurysms, implying a potentially important role
for this macrophage enzyme in this atherosclerotic disease
also.40 Interstitial collagenase
and stromelysin have also been identified in atherosclerotic
coronary arteries.4 41 The factors regulating the
production of these metalloproteinases in atherosclerotic
lesions are not yet known. The earlier finding that TNF- and
interleukin-1ß5 and the present finding that C
pneumoniae and its proteins can increase 92-kDa gelatinase
production by human monocyte–derived macrophages in
vitro, but not that of interstitial collagenase
or stromelysin, reveals differences in the mechanisms regulating the
production of these 3 metalloproteinases. Provided that C
pneumoniae actually leads to activation of macrophages in
atherosclerotic plaques and induces secretion of active 92-kDa
gelatinase by these cells, the ensuing matrix degradation would
increase the susceptibility of the plaques to rupture. This suggests a
mechanism by which C pneumoniae could contribute to the
pathology of atherothrombosis.
An association has been found between infection with C pneumoniae and myocardial infarction. Interestingly, recent clinical findings showed that acute respiratory infection is associated with an increased risk of acute myocardial infarction.42 In a case-control study, previous use of tetracyclines or quinolones, antibiotics that are active against C pneumoniae, was associated with a lower incidence of first-time acute myocardial infarction.43 Both of these findings include the possibility but do not prove that C pneumoniae is associated with myocardial infarction. Besides antimicrobial activity, some other pharmacological mechanisms, ie, suppression of metalloproteinase secretion by tetracyclines,44 could contribute to the reduced risk of acute myocardial infarction. Experience from other antibiotic intervention trials suggests that treatment with azithromycin45 or roxithromycin46 may also improve the prognosis of chronic and acute coronary heart disease. Unfortunately, the mere association of chlamydial infection with myocardial infarction and the results of these antibiotic trials cannot provide us with an answer as to whether the present in vitro observation is of clinical significance. Notably, chlamydial infection without seeding of the organisms in the coronary arteries, by causing systemic inflammation and elevation of C-reactive protein, also increases the risk of acute myocardial infarction.47 Moreover, the current modes of antibiotic treatment are not chlamydia-specific and may also have direct anti-inflammatory effects. However, eradication of C pneumoniae from macrophages in atherosclerotic plaques by treatment with antibiotics appears to be a means of preventing macrophages from secreting matrix-degrading enzymes, and in this way the therapy could also lower the risk of plaque rupture.
|
Acknowledgments |
|---|
The Wihuri Research Institute is maintained by the Jenny and Antti Wihuri Foundation, and this study was partially supported by contract BIO-CT96–0152 of the Biotechnology Program of the Commission of the European Union. We gratefully acknowledge the valuable technical assistance of Anja Ratilainen and Saija Kokkoniemi and Dr Ken Lindstedt for advice in performing the RT-PCR experiments. We also thank Dr Jari Haukka for careful statistical analysis.
Received January 14, 2000; accepted July 27, 2000.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Matrisian LM. The matrix-degrading metalloproteinases. Bioessays. 1992;14:455–463.[Medline] [Order article via Infotrieve]
2.
Okada Y, Takeuchi N, Tomita K, Nakanishi I, Nagase H.
Immunolocalization of matrix metalloproteinase 3 (stromelysin) in
rheumatoid synovioblasts (B cells): correlation with rheumatoid
arthritis. Ann Rheum Dis. 1989;48:645–653.
3. Dean DD, Martel-Pelletier J, Pelletier J-P, Howell DS, Woessner JF. Evidence for metalloproteinase and metalloproteinase inhibitor imbalance in human osteoarthritic cartilage. J Clin Invest. 1989;84:678–685.
4. Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest. 1994;94:2493–2500.
5.
Sarén P, Welgus HG, Kovanen PT. TNF- and
IL-1ß selectively induce expression of 92-kDa gelatinase by human
macrophages. J Immunol. 1996;157:4159–4165.[Abstract]
6. Welgus HG, Campbell EJ, Cury JD, Eisen AZ, Senior RM, Wilhelm SM, Goldberg GI. Neutral metalloproteinases produced by human mononuclear phagocytes: enzyme profile, regulation, and expression during cellular development. J Clin Invest. 1990;86:1496–1502.
7. George SJ. Tissue inhibitors of metalloproteinases and metalloproteinases in atherosclerosis. Curr Opin Lipidol. 1998;9:413–423.[Medline] [Order article via Infotrieve]
8. Kähäri V-M, Saarialho-Kere U. Matrix metalloproteinases in skin. Exp Dermatol. 1997;6:199–213.[Medline] [Order article via Infotrieve]
9. Matrisian LM. Metalloproteinases and their inhibitors in matrix remodeling. Trends Genet. 1990;6:121–125.[Medline] [Order article via Infotrieve]
10. Welgus HG, Campbell EJ, Bar-Shavit Z, Senior RM, Teitelbaum SL. Human alveolar macrophages produce a fibroblast-like collagenase and collagenase inhibitor. J Clin Invest. 1985;76:219–224.
11.
Halpert I, Sires UI, Roby JD, Potter-Perigo S, Wight
TN, Shapiro SD, Welgus HG, Wickline SA, Parks WC. Matrilysin is
expressed by lipid-laden macrophages at sites of potential
rupture in atherosclerotic lesions and localizes to areas of versican
deposition, a proteoglycan substrate for the enzyme. Proc Natl
Acad Sci U S A. 1996;93:9748–9753.
12. Hibbs MS, Hoidal JR, Kang AH. Expression of a metalloproteinase that degrades native type V collagen and denatured collagens by cultured human alveolar macrophages. J Clin Invest. 1987;80:1644–1650.
13.
Shapiro SD, Griffin G, Gilbert DJ, Jenkins NA, Copeland
NG, Welgus HG, Senior RM, Ley TJ. Molecular cloning, chromosomal
localization and bacterial expression of a novel murine
macrophage metalloelastase. J Biol Chem. 1992;267:4664–4671.
14. Denhardt DT, Feng B, Edwards DR, Cocuzzi ET, Malyankar UM. Tissue inhibitor of metalloproteinases (TIMP, aka EPA): structure, control of expression and biological functions. Pharmacol Ther. 1993;59:329–341.[Medline] [Order article via Infotrieve]
15. Lacraz S, Nicod LP, Chicheportiche R, Welgus HG, Dayer J-M. IL-10 inhibits metalloproteinase and stimulates TIMP-1 production in human mononuclear phagocytes. J Clin Invest. 1995;96:2304–2310.
16. Cook PJ, Honeybourne D. Chlamydia pneumoniae. J Antimicr Chemother. 1994;34:859–873.
17. Melgosa MP, Kuo C-C, Campbell LA. Outer membrane complex proteins of Chlamydia pneumoniae. FEMS Microbiol Lett. 1993;112:199–204.
18. Campbell LA, Kuo C-C, Grayston JT. Structural and antigenic analysis of Chlamydia pneumoniae. Infect Immun. 1990;58:93–97.
19.
Mygind P, Christiansen G, Persson K, Birkelund S.
Analysis of the humoral immune response to Chlamydia
outer membrane protein 2. Clin Diagn Lab Immunol. 1998;5:313–318.
20.
Kikuta LC, Puolakkainen M, Kuo C-C, Campbell LA.
Isolation and sequence analysis of the Chlamydia
pneumoniae GroE operon. Infect Immun. 1991;59:4665–4669.
21. Saikku P, Mattila K, Nieminen MS, Huttunen JK, Leinonen M, Ekman M-R, Mäkelä PH, Valtonen V. Serological evidence of an association of a novel Chlamydia, TWAR, with chronic coronary heart disease and acute myocardial infarction. Lancet. 1988;2:983–985.[Medline] [Order article via Infotrieve]
22.
Kuo C-C, Gown AM, Benditt EP, Grayston JT. Detection of
Chlamydia pneumoniae in aortic lesions of
atherosclerosis by immunocytochemical stain.
Arterioscler Thromb. 1993;13:1501–1504.
23.
Kol A, Sukhova GK, Lichtman AH, Libby P. Chlamydial
heat shock protein 60 localizes in human atheroma and
regulates macrophage tumor necrosis factor- and matrix
metalloproteinase expression. Circulation. 1998;98:300–307.
24. Caldwell HD, Kromhout J, Schachter J. Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis. Infect Immun. 1981;31:1161–1176.
25. Kuo C-C, Grayston JT. A sensitive cell line, HL cells, for isolation and propagation of Chlamydia pneumoniae strain TWAR. J Infect Dis. 1990;162:755–758.[Medline] [Order article via Infotrieve]
27. Giambernardi TA, Grant GA, Taylor GP, Hay RJ, Maher VM, McCormick JJ, Klebe RJ. Overview of matrix metalloproteinase expression in cultured human cells. Matrix Biol. 1998;16:483–496.[Medline] [Order article via Infotrieve]
28. Sorger T, Germinario RJ. A direct solubilization procedure for the determination of DNA and protein in cultured fibroblast monolayer. Anal Biochem. 1983;131:254–256.[Medline] [Order article via Infotrieve]
29. Laird NM, Ware JH. Random-effects models for longitudinal data. Biometrics. 1982;38:963–974.[Medline] [Order article via Infotrieve]
30. Sato H, Seiki M. Regulatory mechanism of 92 kDa type IV collagenase gene expression which is associated with invasiveness of tumor cells. Oncogene. 1993;8:395–405.[Medline] [Order article via Infotrieve]
31.
Xu X-P, Meisel SR, Ong JM, Kaul S, Cercek B,
Rajavashisth TB, Sharifi B, Shah PK. Oxidized low-density lipoprotein
regulates matrix metalloproteinase-9 and its tissue
inhibitor in human monocyte-derived macrophages.
Circulation. 1999;99:993–998.
32.
Dechend R, Maass M, Gieffers J, Dietz R, Scheidereit C,
Leutz A, Gulba DC. Chlamydia pneumoniae infection of
vascular smooth muscle and endothelial cells
activates NF-B and induces tissue factor and PAI-1
expression. Circulation. 1999;100:1369–1373.
33. Fisher GJ, Datta SC, Talwar HS, Wang Z-Q, Varani J, Kang S, Voorhees JJ. Molecular basis of sun-induced premature skin ageing and retinoid antagonism. Nature. 1996;379:335–339.[Medline] [Order article via Infotrieve]
34. Godzik KL, O’Brien ER, Wang S-K, Kuo C-C. In vitro susceptibility of human vascular cells to infection with Chlamydia pneumoniae. J Clin Microbiol . 1995;33:2411–2414.
35. Yong EC, Chi EY, Kuo C-C. Differential antimicrobial activity of human mononuclear phagocytes against the human biovars of Chlamydia trachomatis. J Immunol . 1987;139:1297–1302.
36.
Davidson M, Kuo C-C, Middaugh JP, Campbell LA, Wang
S-P, Newman III WP, Finley JC, Grayston JT. Confirmed previous
infection with Chlamydia pneumoniae (TWAR) and its presence
in early coronary atherosclerosis.
Circulation. 1998;98:628–633.
37.
Grayston JT, Kuo C-C, Coulson AS, Campbell LA, Lawrence
RD, Lee MJ, Strandness ED, Wang S-P. Chlamydia pneumoniae
(TWAR) in atherosclerosis of the carotid artery.
Circulation. 1995;92:3397–3400.
38.
Kuo C-C, Grayston JT, Campbell LA, Goo YA, Wissler RW,
Benditt EP. Chlamydia pneumoniae (TWAR) in coronary
arteries of young adults (15–34 years old). Proc Natl Acad Sci
U S A. 1995;92:6911–6914.
39.
Brown DL, Hibbs MS, Kearney M, Loushin C, Isner JM.
Identification of 92-kD gelatinase in human coronary
atherosclerotic lesions: association of active enzyme synthesis with
unstable angina. Circulation. 1994;91:2125–2131.
40. Thompson RW, Holmes DR, Mertens RA, Liao S, Botney MD, Mecham RP, Welgus HG, Parks WC. Production and localization of 92-kilodalton gelatinase in abnormal aortic aneurysms: an elastolytic metalloproteinase expressed by aneurysm-infiltrating macrophages. J Clin Invest. 1995;96:318–326.
41.
Henney AM, Wakeley PR, Davies MJ, Foster K, Hembry R,
Murphy G, Humphries S. Localization of stromelysin gene expression in
atherosclerotic plaques by in situ hybridization. Proc
Natl Acad Sci U S A. 1991;88:8154–8158.
42. Meier CR, Jick SS, Derby LE, Vasilakis C, Jick H. Acute respiratory-tract infections and risk of first-time acute myocardial infarction. Lancet. 1998;351:1467–1471.[Medline] [Order article via Infotrieve]
43.
Meier CR, Derby LE, Jick SS, Vasilakis C, Jick H.
Antibiotics and risk of subsequent first-time acute myocardial
infarction. JAMA. 1999;281:427–431.
44. Duivenvoorden WC, Hirte HW, Singh G. Use of tetracycline as an inhibitor of matrix metalloproteinase activity secreted by human bone-metastasizing cancer cells. Invasion Metastasis. 1997;17:312–322.[Medline] [Order article via Infotrieve]
45.
Gubta S, Leatham EW, Carrington D, Mendall MA, Kaski
JC, Camm AJ. Elevated Chlamydia pneumoniae antibodies,
cardiovascular events, and azithromycin in male
survivors of myocardial infarction. Circulation. 1997;96:404–407.
46.
Gurfinkel E, Bozovich G, Beck E, Testa E, Livellara B,
Mautner B. Treatment with the antibiotic roxithromycin in patients with
acute non-Q-wave coronary syndromes. Eur Heart
J. 1999;20:121–127.
47.
Roivainen M, Viik-Kajander M, Palosuo T, Toivanen P,
Leinonen M, Saikku P, Tenkanen L, Manninen V, Hovi T,
Mänttäri M. Infection, inflammation, and the risk of
coronary heart disease. Circulation. 2000;101:252–257.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||