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Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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are particularly important in
protective immunity against intracellular pathogens, including
chlamydiae, and IL-18 together with IL-12 are strong inducers of
IFN- secretion by CD4 T cells. Because epithelial cells are known to
synthesize IL-18, we investigated the effects of Chlamydia
trachomatis infection of human epithelial cell lines on IL-18
secretion. We confirmed that several human epithelial cell lines
constitutively express pro-IL-18 and that C. trachomatis
infection causes cells to secrete mature IL-18. This was observed for
several different serovars and biovars of C.
trachomatis. Chlamydia-induced secretion of IL-18 from
epithelial cells was regulated at the posttranscriptional level and was
dependent on the activation of caspase-1. IL-1
or other secreted
factor(s) from chlamydia-infected epithelial cells as well as
chlamydial structural component(s) were not involved in inducing IL-18
secretion. Activation of caspase-1 and increased secretion of mature
IL-18 was correlated with chlamydial, but not with host protein
synthesis. In contrast to epithelial cell lines, fibroblast cell lines
constitutively expressed much lower levels of pro-IL-18 and did not
secrete mature IL-18 after chlamydial infection even though caspase-1
was activated. Taken together, the results suggest that a
chlamydia-derived factor(s) is essential for the secretion of mature
IL-18 through caspase-1 activation in infected epithelial
cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3 million cases per year
(1). Chlamydial infection in women can cause salpingitis,
resulting in ectopic pregnancy and tubal infertility, which are the
major public health burdens of this sexually transmitted infection
(1, 2). In addition, in many areas of the developing
world, C. trachomatis ocular infection is
the cause of trachoma, which can result in blindness. Collectively,
genital and ocular C. trachomatis infections are major
causes of morbidity throughout the world.
The epithelial cell layer that lines the various mucosal surfaces of
the body is both a physical barrier against entry of pathogenic
organisms and also the initial cellular target for microbial infection.
Epithelial cells appear to serve as sensitive indicators of infection
by actively initiating an early host defense response through the
secretion of chemokines and proinflammatory cytokines (3, 4). Chemokines of the C-X-C and C-C family and proinflammatory
cytokines such as IL-1, IL-1ß, IL-6, TNF-, and GM-CSF produced
by epithelial cells recruit immune cells to the epithelial/mucosal
surface and help in the activation of macrophages and dendritic cells
and the differentiation of effector lymphocytes (3, 5, 6).
Regulatory cytokines involved in the development of Th1/Th2 responses
such as IL-12, IFN-, and IL-4 are not known to be released from
epithelial cells after microbial infection (5). However,
IL-18, which cooperates with IL-12 in the regulation of Th1 cytokine
responses is known to be expressed in intestinal epithelial cells and
thus may be important in the differentiation of effector T cells to a
Th1 cytokine response pattern (7, 8).
IL-18 was originally identified as IFN--inducing factor and has
structural similarities with the IL-1 family of proteins. Gene
expression and/or protein secretion of IL-18 have been observed in
macrophages (9), dendritic cells (10),
mononuclear cells (11), keratinocytes (12),
osteoblast cells (13), pituitary gland and adrenal
cortical cells (14), astrocytes and microglia
(15), and intestinal epithelial cells (7, 8).
It is also known that both precursor and mature forms of IL-18 exist.
Caspase-1 is an intracellular cysteine protease that is synthesized as
a precursor protein of 45 kDa and which is cleaved to an intermediate
and then to p20 and p10 forms, which are the biologically active forms
of the enzyme. Activated caspase-1 cleaves the IL-18 precursor
(pro-IL-18) into the mature form, and only mature IL-18 is bioactive
(16).
The development of Th1-mediated immune responses is necessary for
protective immunity against a variety of intracellular microbes
including chlamydia (17, 18, 19, 20). IL-18 potentiates
IL-12-dependent Th1 development by enhancing the IFN- production and
NK cell activity (16), suggesting that IL-18 may play a
role in the induction of chlamydia immunity. Because the mucosa is the
initial site of chlamydia infection, we explored the possibility that
chlamydia induces the secretion of IL-18 from epithelial cells. We
found that epithelial cell lines but not fibroblast cell lines
constitutively express pro-IL-18 and secrete mature IL-18 after
chlamydia infection. IL-18 release was delayed until 24 h after
infection, was prevented by inhibitors of chlamydial but not host cell
protein synthesis, and was dependent on the activation of caspase-1.
Our results indicate a role for chlamydia-derived product(s) in the
activation of caspase-1 and subsequent secretion of mature IL-18.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human HeLa cervix epitheloid cell (CCL2), A549 airway
epithelial cell (CCL185), HT-29 colonic epithelial cell (HTB38), MRC-5
lung fibroblast (CCL171), and HT-1018 fibrosarcoma cell (CCL121) were
obtained from American Type Culture Collection (Manassas, VA). Human
2C4 fibroblast was kindly provided by Dr. G. Stark (Cleveland Clinic
Foundation, Cleveland, OH). HeLa, A549, MRC-5, HT-1080, and 2C4 cells
were grown in MEM (Life Technologies, Grand Island, NY) containing 10%
FCS, whereas HT-29 cells were grown in RPMI 1640 (Life Technologies)
with 10% FCS. Penicillin, chloramphenicol, genestein, MG-132,
pyrrolidinedithiocarbamate, N-tosyl-1-phenylalanine
chloromethyl-ketone (TPCK),3
and cycloheximide were purchased from Sigma (St. Louis, MO).
Calphostin C, chelerythrine chloride, herbimycin A, hypericin, PD98059,
SB203580, clasto-lactacystin ß-lactone, and caspase-1 inhibitor II
(Ac-YVAD-CMK) were purchased from Calbiochem (La Jolla, CA). Anti-human
IL-1, IL-1R type I/p80 (IL-1RI) mAbs, and matched isotype Igs were
purchased from R&D Systems (Minneapolis, MN) and PharMingen (San Diego,
CA), respectively. Recombinant human IL-1 and IL-1ß were obtained
from Biological Resource Branch, National Cancer Institute
(Frederick, MD).
Chlamydial strains
C. trachomatis serovars L2, E, F, and K and biovar mouse pneumonitis (MoPn) were propagated in HeLa cell monolayers and purified on discontinuous gradients of Renografin-76 (Squibb Canada, Montreal, Canada) as previously described (21, 22). Purified elementary bodies (EBs) were resuspended in isotonic sucrose-phosphate-glutamate (SPG) buffer and stored at -80°C. The infectivity of purified EBs was titrated by counting chlamydial inclusion-forming units (IFUs) on the monolayer of HeLa cells grown in a 96-well plate. Portions of the purified EBs were heat-inactivated at 75°C for 30 min. For convenience, the number of heat-killed EBs was calculated based on the number of IFUs of the corresponding live EBs before heat treatment.
Infection protocol
Confluent epithelial cell or fibroblast monolayer in a 96-well plate was inoculated with various multiplicities of infection (moi) of EBs (EBs per host cell) in SPG or SPG alone as control. The plate was rocked for 2 h at 37°C to allow attachment and bacterial entry to occur, after which the extracellular bacteria were removed by washing and cells were cultured with growth medium only or in the presence of antibiotics, Ab, or inhibitors for various times as indicated. Supernatants were stored at -80°C until cytokine assay. Conditioned medium was collected from HeLa cells at day 2 after infection with moi of 2 and was stored at -80°C after centrifuging at 13,000 x g for 30 min to remove chlamydia. Samples for Western blot and RT-PCR analysis were obtained similarly, except that cells were grown in a 25-cm2 flask. The infection protocol of mouse macrophage was similar to those of epithelial cells. In brief, caspase-1 knockout (KO) and wild-type (WT) mice (kindly provided by Dr. Winnie Wong, BASF Bioresearch Corporation, Worcester, MA) were i.p. injected with 3% thioglycolate, respectively. On day 3, peritoneal macrophages were obtained and seeded into a 96-well plate. After 2 h in culture, nonattached cells were removed and the plate was inoculated with serovar L2 or biovar MoPn for another 2 h. The supernatants were harvested after 2 days of culture and subjected to mouse IL-18 ELISA.
Cytokine assay
Cytokine levels in culture supernatants were determined by a sandwich ELISA. Ab pairs for human and mouse IL-18 ELISA, which detect IL-18 from either species, were purchased from Research Diagnostics (Flanders, NJ). Capturing and detecting Abs for human IL-8 ELISA were obtained from PharMingen.
Western blot analysis
Epithelial cells or fibroblasts were lysed in radioimmunoprecipitation assay buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 1 µg/ml aprotinin, 10 µg/ml leupeptin, 1 µg/ml pepstatin A, and 1 mM sodium orthovanadate). After centrifugation at 13,000 x g at 4°C for 15 min, 20 µg of supernatant protein from each sample was run on 15% SDS-polyacrylamide gel and then transferred to polyvinylidene difluoride membrane. Blots were preincubated in PBS containing 5% nonfat milk overnight at 4°C and then stained by primary Abs, mouse anti-chlamydia major outer membrane protein (MOMP) mAb (MC22; provided by Dr. G. Zhong, University of Manitoba, Winnipeg, Canada), mouse anti-human IL-18 mAb (Research Diagnostics), or rabbit anti-human caspase-1 Ab (R105; a gift of Dr. D. Miller, Merck Laboratories, Rahway, NJ), respectively. After secondary staining with peroxidase-conjugated goat anti-mouse or rabbit IgG (Jackson ImmunoResearch, West Grove, PA), respectively, enhanced chemiluminescence (Amersham Life Sciences, Arlington Heights, IL) was used to visualize chlamydia MOMP, IL-18, and caspase-1 bands. Recombinant human IL-18 (Research Diagnostics) was used as positive control.
Semiquantitative RT-PCR
Total RNA was extracted from cells using Trizol reagent (Life Technologies Laboratories). For cDNA synthesis, 5 µg of total RNA was reverse transcribed with oligo dT primers and AWV reverse transcriptase (Invitrogen, Carlsbad, CA) and then amplified by human IL-18 or ß-actin specific primers (Canadian Life Technologies, Burlington, Ontario, Canada). PCR mixture contained 2 µl of cDNA, 50 mM Tris-HCl (pH 9.0), 50 mM KCl, 2.5 mM MgCl2, 0.2 mM dNTP, 30 pM of each primer, and 2.5 U Taq polymerase. The PCR condition was previously described, except that the cycle number was reduced to 25 (7, 23). PCR products were electrophoresed on 1% agarose gel and visualized by ethidium bromide staining. PCR products with ß-actin primers were used as internal control on each sample. The sequences of human IL-18 primers were 5'-GCTTGAATCTAAATTATCAGTC-3' (forward) and 5'-GAAGATTCAAATTGCATCTTAT-3' (reverse), resulting in a 279-bp amplified fragment. The sequences of ß-actin primers were 5'-GTGGGGCGCCCCAGGCACCA-3' (forward) and 5'-CTCCTTAATGTCACGCACGATTTC-3' (reverse), resulting in an 880-bp fragment. RT-PCR was further performed in the LightCycler (Roche Molecular Biochemicals, Quebec, Canada) according the instruction of manufacturer. LightCycler-PCR condition was the same as conventional PCR described above, except for the addition of LightCycler-DNA master SYBR green (Roche Molecular Biochemicals) to the reaction mixture. This technique continuously monitors the accumulation of fluorescently labeled PCR product to calculate starting transcript numbers as described by Morrison et al. (24).
Statistical analyses
The data were expressed as mean ± SEM. Student’s t test was used to calculate significance levels between groups with p < 0.05 interpreted as statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first investigated whether chlamydial infection induces the
secretion of IL-18 by epithelial cells. HeLa cells were infected with
C. trachomatis serovar L2 and cultured
for up to 4 days, which is sufficient for at least one cycle of
chlamydial replication and developmental differentiation. The increased
IL-18 secretion from HeLa cells became detectable about 24 h after
infection and remained elevated thereafter (Fig. 1
a). More than 98% of HeLa
cells were seen to contain small inclusion bodies 1 day after infection
with a moi of 1 or higher, suggesting that the enhanced secretion of
IL-18 comes from infected cells rather than as a compensatory effect of
uninfected neighboring cells. Essentially, similar patterns were seen
in experiments using A549 and HT-29 epithelial cells (Fig. 1, b and c). Even when a higher moi (20:1) was used,
the secretion of IL-18 still did not occur until after the first
24 h of infection in all three tested cell lines.
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d, all tested
serovars/biovars enhanced IL-18 release in an inoculum size-dependent
pattern after infection of HeLa cells. Similar results were also
apparent in A549 lung cells and HT-29 colon cells when tested with the
various serovars and biovar of C. trachomatis. Thus, the
IL-18 response of epithelial cells to chlamydial infection appears to
be a shared feature for several different epithelial cell lines and
different C. trachomatis serovars and biovars. Chlamydia-induced IL-18 secretion is regulated at the posttranscriptional level through a caspase-1-dependent mechanism
To study the mechanism(s) by which chlamydia induces IL-18
release, we evaluated IL-18 mRNA and protein expression levels in HeLa
and A549 cells after serovar L2 infection. These
epithelial cells constitutively expressed IL-18 mRNA and protein. After
infection, there was no significant alteration of the IL-18 mRNA or
pro-IL-18 protein expression (Fig. 2,
a and b). LightCycler PCR also confirmed that
there was no significant difference in the relative amounts of IL-18
mRNA between infected and noninfected HeLa cells (Fig. 2d).
Western blot analysis showed that the 18.3-kDa mature form of IL-18
appeared only in infected epithelial cells, whereas both infected and
noninfected epithelial cells contained abundant 24-kDa pro-IL-18 (Fig. 2b). The results indicate that the chlamydia induction of
IL-18 secretion occurred at the posttranscription level.
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a). We investigated the role
of activated capase-1 on the chlamydia-induced IL-18 release from
infected epithelial cells by using a cell-permeable, caspase-1-specific
inhibitor, the peptide Ac-YVAD-CMK. As expected, inhibition experiments
showed that infected HeLa cells had nondetectable expression of mature
IL-18 in the presence of Ac-YVAD-CMK, whereas there was no significant
inhibitory effect on the expression of pro-IL-18 (Fig. 2b).
ELISA results also showed significant dose-dependent inhibition by
Ac-YVAD-CMK on IL-18 release from infected epithelial cells (Fig. 3b). Therefore, the results indicated that the increased
release of IL-18 was caspase-1-dependent and largely consisted of
mature IL-18. The inhibition was specific for IL-18 release because
there was no inhibitory effect by the peptide on IL-8 secretion (Fig. 3b). To confirm that caspase-1 is necessary for
chlamydia-induced IL-18 secretion, we infected peritoneal macrophages
from caspase-1 KO mice and WT mice with serovar
L2 or biovar MoPn, respectively. As shown in Fig. 3c, no increased secretion of IL-18 occurred from
chlamydia-infected macrophages lacking caspase-1. Collectively, these
results confirm that caspase-1 activity is essential for
chlamydia-induced IL-18 secretion from host cells and that chlamydia
activates caspase-1 in host cell.
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or other secreted factor(s) from infected
epithelial cells
During in vivo or in vitro culture, cytokine response to an
initial stimulus can be influenced by other cytokine(s) or factor(s) in
the local environment or culture system. We characterized factors in
the culture that may elicit IL-18 secretion after chlamydia infection.
Previous studies by Rasmussen et al. (22) showed that Ab
to IL-1 blocked most of the secretion of IL-8, IL-6, GM-CSF, and
growth-regulated oncogene- from chlamydia-infected HeLa cells,
indicating that IL-1 is an upstream factor regulating the release of
proinflammatory cytokines and chemokines from infected cells.
Therefore, we explored the possibility that the induction of IL-18 in
chlamydia-infected epithelial cells may also be secondary to the
increased secretion of IL-1. We found that IL-1 or IL-1RI Abs did
not block the secretion of IL-18 from the infected HeLa cells (Fig. 4). Furthermore, the addition of
exogenous IL-1 did not significantly increase IL-18 secretion from
the three tested epithelial cell lines (Fig. 4 and data not shown). In
contrast, exogenous IL-1 did significantly increase IL-8 secretion
from HeLa cells in a dose-dependent pattern (e.g., 10 ng/ml IL-1
treated, 1.4 ± 0.2 ng/ml vs control (0.1 ± 0.03 ng/ml);
data are mean ± SEM of triplicate cultures; the same supernatants
were subjected to the IL-18 assay for the above results). The effect of
IL-1 on the IL-8 secretion was completely blocked by either IL-1
or IL-1RI Ab, indicating that epithelial cells are IL-1
responsive. Therefore, we conclude that the increased IL-18
secretion from chlamydia-infected epithelial cells is not mediated via
IL-1. Similar conclusions were also reached when using IL-1ß
instead of IL-1 (data not shown).
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Increased IL-18 secretion from chlamydial-infected epithelial cells is dependent on chlamydial, but not host protein(s) synthesis
To elucidate the mechanism of chlamydia-induced IL-18 secretion in
human epithelial cell lines, we evaluated the effects of chlamydial
components on the secretion of IL-18. Live C. trachomatis
serovar L2 significantly increased IL-18
secretion, which was detected around 24 h postinfection and peaked
at day 3 (Figs. 1 and 3–5). Heat-killed
chlamydia did not induce IL-18 secretion even when tested for up to 3
days postculture (Fig. 5). Use of higher moi of heat-killed EBs, e.g.,
10 IFU/cell, for up to 3 days also failed to induce IL-18 secretion by
HeLa cell or A549 cell (data not shown). This suggested that chlamydial
LPS or other structural components of the bacterial cell are not
sufficient to elicit an increase in IL-18 secretion from epithelial
cells.
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, serovar L2 infection of
HeLa cells was followed by IL-18 secretion, and this was paralleled
with expression of chlamydial MOMP, suggesting that IL-18 release was
dependent on chlamydia protein synthesis or/and growth. Indeed, serovar
L2-induced IL-18 secretion could be prevented by
chloramphenicol (Fig. 6a and
data not shown). The effect of chloramphenicol is likely attributed to
inhibition of chlamydia protein synthesis (25) because
penicillin, which blocks reticulate body differentiation to EB
(26), only partly affected the IL-18 response (Fig. 6a). Cycloheximide, which is a eukaryotic but not
prokaryotic protein synthesis inhibitor, had no affect on mature IL-18
production after chlamydia infection (Fig. 6b). Therefore,
the data indicate that chlamydia growth and protein synthesis during
the first 24 h of infection are required for increased IL-18
secretion from infected epithelial cells.
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B as described in Materials and Methods. IL-18 levels
were measured after 2 days of culture. None of the tested protein
kinase C, mitogen-activated protein kinase, protein tyrosine kinase, or
NF-B inhibitors significantly reduced chlamydia induction of IL-18
secretion from epithelial cells. Two different proteasome inhibitors,
TPCK and lactacystin, decreased chlamydia-induced IL-18 release in a
dose-dependent pattern (Figs. 6b and
7). Neither proteasome inhibitor
inhibited chlamydia growth in epithelial cells as determined by the
chlamydia MOMP expression levels (Fig. 6c). The results
indicated that the proteasome may be involved in the activation of
caspase-1. Lack of IL-18 secretion by fibroblast cells after C. trachomatis infection
Although epithelial cell lines respond to C.
trachomatis infection with IL-18 secretion, this response was not
demonstrated by infected MRC-5, 2C4, and HT-1080 fibroblast cell
lines, even though chlamydia replicated very well in these cell lines
(Fig. 6c) and caspase-1 activation occurred (data not
shown). Western blot analysis showed that fibroblast cell lines
contained low levels of pro-IL-18 protein (Fig. 6b), which
corresponded with very low levels of mRNA transcription as measured by
LightCycler-PCR analysis (Fig. 8). The
results indicated that IL-18 secretion from a cell type depends not
only on the chlamydia-dependent activation of caspase-1 but also on the
sufficient levels of constitutive pro-IL-18 within the host
cell.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several reports have suggested that proinflammatory cytokines can be released from infected epithelial cells following host cell lysis after infection (27, 28, 29). Chlamydiae replicate in a specialized vacuole in the host cell cytoplasm and induce host cell lysis at the end of its developmental cycle (30). However, the process of cell lysis after chlamydia infection is relatively slow (especially in the absence of cycloheximide), requires several days to occur, and depends on the inoculum size. In the present study, flow cytometry analysis showed that there was more than 95% viable cells in both infected and noninfected HeLa cells using propidium iodide staining at 3 days of culture when a moi below 2 was used (data not shown). Furthermore, we also found no significant difference of lactate dehydrogenase levels in culture supernatants between infected or noninfected HeLa cells (data not shown). Thus, the enhanced secretion of IL-18 from chlamydia-infected epithelial cell lines was not likely passively released by cell lysis.
IL-18 is a new member of the IL-1 family of cytokines, which includes
IL-1, IL-1ß, and IL-1R antagonist, all of which have pleiotropic
activities involved in immune and inflammatory responses (16, 31). Little is known of how a microbial pathogen might regulate
IL-18 secretion from epithelial cells. IL-1 is an upstream cytokine
that regulates the de novo production of other proinflammatory
cytokines and chemokines, such as IL-8, IL-6, GM-CSF, growth-regulated
oncogene-, and TNF-, from epithelial cells or endothelial cells
infected with microbial pathogens such as C. trachomatis
(22), Rickettsia conorii (28),
Entamoeba histolytica (27), Toxoplasma
gondii (29), and respiratory syncytial virus
(32). Our observations demonstrate that increased IL-18
secretion in epithelial cells after C. trachomatis
infection is not mediated by IL-1 or other secreted factor(s),
indicating that IL-18 secretion from epithelial cells infected with
chlamydia differs from that regulating the secretion of IL-8, IL-6,
GM-CSF, TNF-, and other proinflammatory cytokines or chemokines.
Caspase-1 is a cysteine protease and proteolytically processes inactive
precursors of IL-18 and IL-1ß to enzymatically active forms. In this
paper we report that chlamydia infection causes the activation of
caspase-1 and that this is correlated with the release of mature IL-18
from epithelial cells. The formation of mature IL-18 was inhibited by a
caspase-1-specific inhibitor in a dose-dependent pattern and did not
occur when macrophages from caspase-1 KO mice were infected by
chlamydia. In aggregate, these data indicate that activation of
caspase-1 is essential for chlamydia induction of IL-18 secretion.
However, the activation of caspase-1 does not appear to be the only
mechanism required for the chlamydia-induced secretion of IL-18 because
similarly infected fibroblast cells did not secrete detectable levels
of IL-18 despite the activation of caspase-1. Other studies have
suggested that the Shigella invasion plasmid Ag B and
Salmonella invasion protein B directly bind to caspase-1 and
trigger hydrolysis of this cysteine protease into its bioactive forms,
which then cleave pro-IL-1ß and trigger host cell apoptosis
(33, 34). Although little is known about the subcellular
sites and detailed mechanisms responsible for activation of caspase-1
(35, 36, 37), we observed that TPCK and lactacystin, two
structurally different but specific inhibitors of proteasomes,
significantly inhibited IL-18 production induced by chlamydia
infection. These results suggest that the proteasome may be involved in
caspase-1 activation as induced by C. trachomatis infection.
Fig. 6 shows that chlamydia, but not host protein synthesis, is
required for the induction of IL-18 secretion during infection. Thus,
it may be that chlamydia-dependent, proteasome-like activity activates
caspase-1. This is analogous to what has been hypothesized for the
inactivation of the transcription factor upstream stimulatory factor
during C. trachomatis infection (38).
It is of interest to note that chlamydia alters a variety of host cell
responses, including proinflammatory cytokine and chemokine secretion
(22), inhibition of apoptosis (39), and
inhibition of IFN--inducible MHC class I and II expression
(38, 40). Chlamydia-induced immune evasion mechanisms may
allow the microbe to escape immune surveillance, especially at early
stages of infection. The selective advantage of IL-18 induction to
chlamydia survival is not apparent because IL-18 should help mobilize
Th1 immune responses that eliminate chlamydia infection from the host.
Rather, the activation of caspase-1 and IL-18 secretion may be
secondary to chlamydia-directed alterations of the proteasome and its
down-regulation of MHC class I and class II expression (38, 40), which might be the selectively favored immune evasion
strategy.
Genome sequencing of chlamydiae has determined that the organism encodes the structural components for a complete type III secretion apparatus (41, 42, 43). Type III injectosomes may provide the route by which chlamydia-derived proteins enter the host cell cytoplasm, similar to that exploited by Shigella flexneri and Salmonella typhimurium (44), and modulate cellular responses such as those involved in the activation of caspase-1 and secretion of IL-18. We are currently searching for type III effector protein(s) such as those that activate caspase-1.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert C. Brunham, University of British Columbia and British Columbia Center for Disease Control, 2077-655 West 12th Avenue, Vancouver, British Columbia V5Z 4R4, Canada.
3 Abbreviations used in this paper: TPCK, N-tosyl-1-phenylalanine chloromethyl-ketone; IL-1RI, IL-1R type I/p80; MoPn, mouse pneumonitis; EB, elementary body; SPG, sucrose-phosphate-glutamate; IFU, inclusion-forming units; moi, multiplicity of infection; KO, knockout; WT, wild type; MOMP, major outer membrane protein.
Received for publication February 3, 2000. Accepted for publication May 16, 2000.
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A. Gervassi, M. R. Alderson, R. Suchland, J. F. Maisonneuve, K. H. Grabstein, and P. Probst Differential Regulation of Inflammatory Cytokine Secretion by Human Dendritic Cells upon Chlamydia trachomatis Infection Infect. Immun., December 1, 2004; 72(12): 7231 - 7239. [Abstract] [Full Text] [PDF]
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H.-P. Raue, J. D. Brien, E. Hammarlund, and M. K. Slifka Activation of Virus-Specific CD8+ T Cells by Lipopolysaccharide-Induced IL-12 and IL-18 J. Immunol., December 1, 2004; 173(11): 6873 - 6881. [Abstract] [Full Text] [PDF]
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 N. Ledee-Bataille, F. Olivennes, J. Kadoch, S. Dubanchet, N. Frydman, G. Chaouat, and R. Frydman Detectable levels of interleukin-18 in uterine luminal secretions at oocyte retrieval predict failure of the embryo transfer Hum. Reprod., September 1, 2004; 19(9): 1968 - 1973. [Abstract] [Full Text] [PDF]
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M. G. Netea, B. J. Kullberg, L. E. H. Jacobs, T. J. G. Verver-Jansen, J. van der Ven-Jongekrijg, J. M. D. Galama, A. F. H. Stalenhoef, C. A. Dinarello, and J. W. M. Van der Meer Chlamydia pneumoniae Stimulates IFN-{gamma} Synthesis through MyD88-Dependent, TLR2- and TLR4-Independent Induction of IL-18 Release J. Immunol., July 15, 2004; 173(2): 1477 - 1482. [Abstract] [Full Text] [PDF]
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R. M. Johnson Murine Oviduct Epithelial Cell Cytokine Responses to Chlamydia muridarum Infection Include Interleukin-12-p70 Secretion Infect. Immun., July 1, 2004; 72(7): 3951 - 3960. [Abstract] [Full Text] [PDF]
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W. Greene, Y. Xiao, Y. Huang, G. McClarty, and G. Zhong Chlamydia-Infected Cells Continue To Undergo Mitosis and Resist Induction of Apoptosis Infect. Immun., January 1, 2004; 72(1): 451 - 460. [Abstract] [Full Text] [PDF]
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C. Obregon, D. Dreher, M. Kok, L. Cochand, G. S. Kiama, and L. P. Nicod Human Alveolar Macrophages Infected by Virulent Bacteria Expressing SipB Are a Major Source of Active Interleukin-18 Infect. Immun., August 1, 2003; 71(8): 4382 - 4388. [Abstract] [Full Text] [PDF]
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 J.-L. Perfettini, D. M. Ojcius, C. W. Andrews Jr., S. J. Korsmeyer, R. G. Rank, and T. Darville Role of Proapoptotic BAX in Propagation of Chlamydia muridarum (the Mouse Pneumonitis Strain of Chlamydia trachomatis) and the Host Inflammatory Response J. Biol. Chem., March 7, 2003; 278(11): 9496 - 9502. [Abstract] [Full Text] [PDF]
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H. Lu, Z. Xing, and R. C. Brunham GM-CSF Transgene-Based Adjuvant Allows the Establishment of Protective Mucosal Immunity Following Vaccination with Inactivated Chlamydia trachomatis J. Immunol., December 1, 2002; 169(11): 6324 - 6331. [Abstract] [Full Text] [PDF]
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S. Dessus-Babus, T. L. Darville, F. P. Cuozzo, K. Ferguson, and P. B. Wyrick Differences in Innate Immune Responses (In Vitro) to HeLa Cells Infected with Nondisseminating Serovar E and Disseminating Serovar L2 of Chlamydia trachomatis Infect. Immun., June 1, 2002; 70(6): 3234 - 3248. [Abstract] [Full Text] [PDF]
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X. Huang, S. A. McClellan, R. P. Barrett, and L. D. Hazlett IL-18 Contributes to Host Resistance Against Infection with Pseudomonas aeruginosa Through Induction of IFN-{gamma} Production J. Immunol., June 1, 2002; 168(11): 5756 - 5763. [Abstract] [Full Text] [PDF]
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 V M Salvati, T T MacDonald, M Bajaj-Elliott, M Borrelli, A Staiano, S Auricchio, R Troncone, and G Monteleone Interleukin 18 and associated markers of T helper cell type 1 activity in coeliac disease Gut, February 1, 2002; 50(2): 186 - 190. [Abstract] [Full Text] [PDF]
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M. S. Pate, S. R. Hedges, D. A. Sibley, M. W. Russell, E. W. Hook III, and J. Mestecky Urethral Cytokine and Immune Responses in Chlamydia trachomatis-Infected Males Infect. Immun., November 1, 2001; 69(11): 7178 - 7181. [Abstract] [Full Text] [PDF]
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 H. Helmby, K. Takeda, S. Akira, and R. K. Grencis Interleukin (IL)-18 Promotes the Development of Chronic Gastrointestinal Helminth Infection by Downregulating IL-13 J. Exp. Med., August 6, 2001; 194(3): 355 - 364. [Abstract] [Full Text] [PDF]
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 O. Yoshino, Y. Osuga, K. Koga, O. Tsutsumi, T. Yano, T. Fujii, K. Kugu, M. Momoeda, T. Fujiwara, K. Tomita, et al. Evidence for the expression of interleukin (IL)-18, IL-18 receptor and IL-18 binding protein in the human endometrium Mol. Hum. Reprod., July 1, 2001; 7(7): 649 - 654. [Abstract] [Full Text] [PDF]
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R. Vankayalapati, B. Wizel, D. L. Lakey, Y. Zhang, K. A. Coffee, D. E. Griffith, and P. F. Barnes T Cells Enhance Production of IL-18 by Monocytes in Response to an Intracellular Pathogen J. Immunol., June 1, 2001; 166(11): 6749 - 6753. [Abstract] [Full Text] [PDF]
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), 1 (
), or 0.1 (
) or with SPG buffer as control (
). IL-18
levels of culture supernatants from HeLa cells (a), A549
cells (b), and HT-29 cells (c) were
determined by ELISA. Similarly, HeLa cells were infected by other
common serovars/biovars, and the supernatants were harvested at 36
h (MoPn and serovar F) or 72 h (serovars E and K) for the IL-18
assay (d). Data are expressed as mean ± SEM of
triplicate cultures from four independent experiments.
