From the Université Paris 7, Institut Pasteur,
Unité de Biologie Moléculaire du Gène, INSERM U277,
Paris, France, ** Sacred Heart Medical Center, Department of
Laboratory Medicine, Spokane, Washington 99220,
Howard Hughes Medical Institute,
Departments of Pathology and Medicine, Harvard Medical School,
Dana-Farber Cancer Institute, Boston, Massachusetts 02115, and the
§§ Department of Microbiology and
Immunology, University of Arkansas for Medical Sciences,
Little Rock, Arkansas 72205
Received for publication, November 4, 2002, and in revised form, December 31, 2002
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ABSTRACT |
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The BCL-2 family member BAX plays a critical role
in regulating apoptosis. Surprisingly, bax-deficient
mice display limited phenotypic abnormalities. Here we investigate the
effect of BAX on infection by the sexually transmitted pathogen,
Chlamydia muridarum (the mouse pneumonitis strain of
Chlamydia trachomatis). Bax Chlamydia species provoke serious infections of humans
and animals worldwide, despite extensive work to better characterize the biology of the infection and develop effective vaccines (1-3). It
is estimated that over 600 million persons are infected with Chlamydia trachomatis, whose strains include the most common
sexually transmitted bacterial pathogen (4) as well as causative agents of conjunctivitis and trachoma. There are an estimated 4 million new
cases annually of genital C. trachomatis infections of the male and female within the United States (5). In women, the most common
consequence of chlamydial genital infection is salpingitis, which can
lead to tubal obstruction and infertility (2).
An important element in the design of a vaccine for the prevention or
control of chlamydial infections is a complete understanding of the
immune response to infection. Little is known about the pathogenesis of
human chlamydial infections, and most of our knowledge of acute
infection has been obtained from animal models such as the mouse model
with Chlamydia muridarum (the mouse pneumonitis (MoPn)1 strain of C. trachomatis) (6, 7) and the guinea pig model with the
Chlamydia psittaci guinea pig inclusion
conjunctivitis strain (8). In controlled studies in guinea pigs and
mice (9-11), bacteria are initially detected in the cervical
epithelium, but the pathology ascends in most animals to the
endometrium and the oviducts within 7-9 days after intravaginal
inoculation. Most of the damage due to Chlamydia is not due
to the infection itself but to the inflammation and fibrosis that
follow the infection (2).
Polymorphonuclear leukocytes are typically observed in the cervix as
early as 2 days after infection, and acute inflammation in the uterine
horns and oviducts follows within 5-7 days (2). A number of
inflammatory mediators are present during infection, including
interleukin-1 (IL-1) and tumor necrosis factor (TNF- Contrasting with the epidemiological and pathological diversity of
Chlamydia infections is the relative uniformity of the chlamydial infectious process. All Chlamydia sp. are thought
to enter into, survive, and multiply within mucosal epithelial cells by
conserved mechanisms involving a unique obligate intracellular developmental cycle, consisting of two phases (16). The extracellular form of Chlamydia, the elementary body (EB), is infectious
and is thought to be metabolically inert. The EB are internalized into
host epithelial cells into small vacuoles resembling endosomes, most of
which avoid fusion with host cell lysosomes. The EB differentiate within the entry vacuole into metabolically active reticulate bodies,
which are presumably noninfectious (17). The reticulate bodies
proliferate within the same membrane-bound vacuole and, after several
divisions, differentiate back into EB. After 2-3 days, the EB are
released from the infected cell through unknown mechanisms and begin a
new cycle of infection (16, 17).
This biphasic developmental cycle allows for multiple sites of
communication between the chlamydial pathogen and the host cell, many
of which probably play a significant role in the pathogen-host cell
relationship and thus strongly impact the outcome of the infection. An
example of such a communication are the chlamydial signals that block
and then later induce apoptosis of the host cell. Like mycobacteria,
Cryptosporidium parvum, and the herpes virus (18-21),
Chlamydia strains protect infected cells during early stages
of the infection against apoptosis due to external stimuli (22-25) and
induce apoptosis of the host cell during later stages of the infection
cycle (26-29). The resistance to cell death may account for the
observation that Fas- and perforin-dependent killer
lymphocytes are not able to clear the infection in mice (30).
Conversely, we had proposed that apoptosis due to the infection may be
used by the chlamydiae to exit from infected cells and propagate within
the host (29).
In mammalian cells, many of the morphological and biochemical features
of apoptosis are due to activation of caspases, which can be initiated
through engagement of cell surface receptors such as Fas (31) or
following release from mitochondria of cytochrome c, which
associates with the apoptosis regulator Apaf-1 and thereby activates
caspase-9, which in turn activates caspase-3 (32). Both pathways are
regulated by the BCL-2 family of proteins, which consists of
antiapoptotic factors, such as BCL-2 and BCL-xL, and proapoptotic proteins, such as BAX and BAK (33). BCL-2 proteins prevent
apoptosis by preventing the release of cytochrome c from mitochondria, whereas BAX stimulates release of cytochrome c
(34, 35). Nonetheless, caspase activation is not required for all types
of cell death (36-38), and overexpression of BAX or BAK induces cell
death without the involvement of caspases (37, 39), suggesting that
factors other than caspases can also mediate apoptosis.
Apoptosis of Chlamydia-infected cells triggered with
external ligands is blocked through inhibition of cytochrome
c release and caspase-3 activation (22), whereas apoptosis
induced by the infection itself is independent of known caspases (28,
29). We have found that BAX is activated and translocates from the cytosol to mitochondria in infected cells, and inhibition of BAX by
overexpression of BAX inhibitor-1 or BCL-2 inhibits
Chlamydia-induced apoptosis (28). Caspase-1 is not thought
to be involved in apoptosis (40), except when targeted specifically by
bacterial products secreted by Shigella flexneri or
Salmonella sp. (41, 42). Nonetheless, caspase-1 is required
for maturation and secretion of IL-1 The preferential target tissue of sexually transmitted chlamydial
infections in females is the columnar epithelium of the cervix (2, 17),
but monocytes and macrophages can also be infected (44) and may aid in
disseminating the infection by certain serovars of
Chlamydia. Since macrophages undergoing apoptosis secrete
IL-1 (45), it is conceivable that apoptosis of these cells during
Chlamydia infection may contribute to the inflammatory response. Conversely, cytokines such as TNF- Since BAX is activated during infection of an epithelial cell line
in vitro (28), we here used Bax-deficient cells
to evaluate the role of BAX in Chlamydia-induced apoptosis
and to investigate the effect of BAX-dependent apoptosis on
the yield of chlamydiae obtained from at least two infection cycles
in vitro. The availability of Bax-deficient mice
also allowed us to confirm a role for BAX during genital tract
infection in vivo and to measure the host inflammatory
response during infection of wild-type and Bax-deficient mice.
Cells and Bacteria--
The mouse pneumonitis agent (MoPn) of
C. trachomatis (C. muridarum) was from
the ATCC (Manassas, VA). Bacteria were prepared, and cells were
infected as previously described (29). The
Bax+/+ (wild type),
Bax Analysis of Cell Death--
Murine embryonic fibroblasts were
infected at a multiplicity of infection of 0.5. Cell death was measured
by cytofluorimetry by staining detergent-permeabilized cells with PI
(29, 49) or by double-staining unpermeabilized cells with PI and
annexin V (48). Both adherent cells and cells in suspension were
collected for analysis.
Effect of BAX on Infectious Activity of
Chlamydia--
Subconfluent Bax+/+ and
Bax Animal Infections--
Female Bax+/+ and
Bax
All mice were given food ad libitum and maintained in
environmentally controlled rooms with a 12/12-h light/dark cycle. All animal studies were approved by the University of Arkansas Medical Sciences' Institutional Animal Care and Use Committee.
Statistics--
Statistical comparisons between the groups of
mice for level of infection, antibody production, and cytokine
production over the course of infection were made by a two-factor (days
and murine strain) analysis of variance with the post
hoc Tukey test as a multiple-comparison procedure. The
Wilcoxon rank sum test was used to compare the duration of infection in
the respective groups over time. One-way analysis of variance on ranks
was used to determine differences in inflammatory cell populations
among the groups. All experiments were repeated at least once.
Effect of BAX on Host Cell Death in Vitro--
We have previously
shown that BAX is activated in cells infected with Chlamydia
(28). The effect of BAX activation on Chlamydia-induced apoptosis was therefore determined by infecting normal
(Bax+/+) and Bax-deficient cells. The
infection led to a high level of apoptosis in
Bax+/+ cells, which was observed after 1 day of
infection (Fig. 1A). At the
same multiplicity of infection, the Bax deficiency resulted in a nearly 2-fold inhibition of apoptosis during infection (Fig. 1A), suggesting that this pathway of apoptosis requires, at
least partially, BAX activation.
Engagement of surface death receptors such as Fas or TNFR1 results in
cleavage of the BCL-2 family member BID, which triggers the
oligomerization of proapoptotic family members BAK and BAX, leading to
cell death (47). To determine whether BID cleavage may be required for
BAX activation in infected cells, Bid+/+ and
Bid
Cells that are prevented from dying through apoptosis still manage to
die, but they often succumb later, dying through necrosis (53-55). To
determine quantitatively whether any infected cells may be necrotic,
cells were infected for 2 days, and necrosis was measured by
double-labeling the cells with PI and annexin V, which binds to
phosphatidylserine (PS) that becomes exposed on the surface of dying
cells. Cells labeled only with annexin V are considered to be
apoptotic, whereas cells labeled only with PI, which have thus lost
their plasma membrane integrity, are necrotic; cells labeled with both
PI and annexin V are either necrotic or late apoptotic (48). Evaluation
by cytofluorimetry showed that the Bax Effect of BAX on Bacterial Yield in Vitro--
In order to
distinguish between the possibility that apoptosis may be used by the
bacteria to escape from the infected host cell, rather than by the host
cell to eliminate bacteria, Bax+/+ and
Bax Effect of BAX on Bacterial Propagation during Genital Tract
Infection--
To confirm whether apoptosis has an effect on the yield
of infectious bacteria in vivo, the infection was repeated
with Bax+/+ and Bax Effect of BAX on Cytokine Secretion during Genital Tract
Infection--
Prior studies in our laboratory have shown that murine
chlamydial genital tract infection induces strong production of the proinflammatory cytokine, TNF- Pathology Associated with Bax Deficiency of Infected
Mice--
Histopathological and immunohistochemical examination of
genital tract tissues from mice sacrificed on day 7 of primary
infection revealed that the early inflammatory response was of similar
quality and quantity in Bax+/+ and
Bax Acquired Immunity in Infected Wild-type and
Bax-deficient Mice--
The acquired immune response, as determined by
antibody titers in serum and by resistance to reinfection, was similar
in Bax We here show that Bax-deficient cells are more
resistant to Chlamydia-induced apoptosis than wild-type
cells. A biological role for BAX activation is suggested by the
observation that the yield of chlamydiae after two infection cycles
decreases in Bax-deficient cells compared with wild-type
cells. BAX could therefore contribute to exit of chlamydiae from
infected cells before initiation of a new infection cycle. The fact
that C. muridarum infection of the genital tract disappears
more rapidly in Bax Apoptotic cells and apoptotic bodies released from dying cells in
vivo are cleared by professional scavengers such as macrophages, which express surface receptors that recognize apoptotic bodies and
cells (62). Thus, PS exposed on the surface of dying cells interacts
with PS receptors on human or murine macrophages, leading to
phagocytosis of the corpses. However, the PS receptor is also expressed
on the surface of fibroblasts and epithelial cell lines, including HeLa
(derived from a carcinoma of the cervix) (56), and ubiquitously
expressed molecules such as lectins or integrins could also participate
in internalization of apoptotic bodies (63). Since an antibody against
the PS receptor can block phagocytosis of apoptotic cells by
fibroblasts and mammary epithelial cells (56), we propose that the PS
receptor and/or similar receptors may be used to internalize
Chlamydia-containing apoptotic cells and bodies by
neighboring epithelial cells in the genital tract, thus beginning a new
round of infection.
Despite the faster clearance of bacteria in
Bax Most of the pathological damage observed during Chlamydia
infection is thought to be due to the inflammatory response rather than
to the microorganism itself (2, 8). The higher incidence of
granulomatous nodules in the Bax Disordered cell death has been previously shown to have an impact on
the immune system and human disease. Thus, reduced cell death and
defective clearance of apoptotic material are thought to lead to
autoimmune diseases, and macrophages secrete proinflammatory mediators
following ingestion of cells undergoing secondary necrosis but not
after ingestion of intact apoptotic cells (62, 81, 82). We find that
defects within the core apoptotic program also lead to immunopathology.
Whereas these diseases may share the common feature that more cells
undergo necrosis when apoptosis is blocked, it is also conceivable that
their pathogenesis may be multifactorial. However, they all demonstrate
clearly that blocking the signaling pathways associated with apoptosis
has consequences for antigens and infectious agents that are normally packaged into apoptotic bodies, with striking effects on host pathology.
/
cells are relatively resistant to Chlamydia-induced
apoptosis, and fewer bacteria are recovered after two infection cycles
from Bax
/
cells than from wild-type cells.
These results suggest that BAX-dependent apoptosis may be
used to initiate a new round of infection, most likely by releasing
Chlamydia-containing apoptotic bodies from infected cells
that could be internalized by neighboring uninfected cells.
Nonetheless, infected Bax
/
cells die
through necrosis, which is normally associated with inflammation, more
often than infected wild-type cells. These studies were confirmed in
mice infected intravaginally with C. muridarum; since the
infection disappears more quickly from Bax
/
mice than from wild-type mice, secretion of proinflammatory cytokines is increased in Bax
/
mice, and large
granulomas are present in the genital tract of Bax
/
mice. Taken together, these data
suggest that chlamydia-induced apoptosis via BAX contributes to
bacterial propagation and decreases inflammation. Bax
deficiency results in lower infection and an increased inflammatory
cytokine response associated with more severe pathology.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
), which have
been detected in the Fallopian tubes from humans infected with C. trachomatis (12) and in secretions from
Chlamydia-infected mice and guinea pigs (13-15). TNF-
and other inflammatory cytokines may aid in eradicating
Chlamydia infection but also may promote long term tissue
damage (14).
and IL-18, and caspase-1 is
activated during Chlamydia infection of monocytes and
epithelial cells (29, 43).
are able to induce apoptosis of some target cells (46), suggesting that the inflammation following Chlamydia infection may also directly trigger apoptosis.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
, Bid+/+, and
Bid
/
murine embryonic fibroblasts were
described (47). All other cells and materials were described (28,
48).
/
cells were infected at a multiplicity
of infection of 0.1, and a 10-fold excess of uninfected
Bax+/+ cells was added after 24 h of
infection. After an additional 2 days of infection, the cells and
supernatant were centrifuged for 60 min at 12,000 rpm in a Sorvall type
GSA rotor. The pellet was freeze-thawed three times and sonicated for
10 min in a bath sonicator at 4 °C to break cells and dissociate
aggregates, giving the final suspension of chlamydiae used to measure
bacterial yield. Serial dilutions of the chlamydial preparation were
used to infect HeLa cells on cover slips for 48 h, and the
chlamydial vacuoles were revealed with fluorescein
isothiocyanate-conjugated anti-Chlamydia monoclonal
antibody, as described (29). Samples were examined with a Zeiss
fluorescence microscope attached to a cooled CCD camera. At least 10 separate fields containing an average of 200-300 HeLa cells were
counted per sample, and the experiment was repeated on three separate occasions.
/
mice on a C57BL/6 background (Jackson
Laboratories, Bar Harbor, MA) were infected intravaginally with
107 inclusion-forming units of C. muridarum. The
course of infection was monitored by periodic cervico-vaginal swabbing
of individual animals (50). Chlamydiae were isolated from swabs in
tissue culture according to standard methods, and inclusions were
visualized and enumerated by immunofluorescence (51). Results are
expressed as mean and S.E. of inclusion-forming units per ml.
Experiments were repeated once, and there were five animals per
experimental group. Groups of mice were sacrificed at 7 and 24 days
after primary infection or followed through day 70 and administered a
challenge infection with 107 inclusion-forming units of
MoPn on day 90, 7 days post-depo-provera treatment. Histopathology and
cytokine secretion measurements were performed as described (50).
Staining of cell surface antigens and qualitative evaluation of cell
populations were performed as described by Morrison and Morrison
(52). Vaginal secretions were assayed individually for cytokine or
chemokine activity by enzyme-linked immunosorbent assay using
commercial kits (R&D Systems, Minneapolis, MN). Antibody responses were
measured in sera from mice and assayed by enzyme-linked immunosorbent
assay as described (14).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of BAX on apoptosis and bacterial
production in vitro. A, apoptosis of wild-type,
Bax /
cells, and
Bid
/
cells. Cells were infected with
C. muridarum for 2 days, and apoptosis of PI-labeled
detergent-permeabilized cells was measured by cytofluorimetry (see
"Experimental Procedures"). Black bar,
spontaneous apoptosis of uninfected cells; white
bar, apoptosis of infected cells. B, necrosis of
infected cells. Bax+/+ and
Bax
/
cells were infected for 48 h.
Necrosis and apoptosis were quantified by double-labeling
unpermeabilized cells incubated with PI and annexin V (see
"Experimental Procedures"). The numbers in each
quadrant refer to the percentage of cells in each quadrant.
C, chlamydial production after at least two infection
cycles. Subconfluent Bax+/+ and
Bax
/
cells were infected at a multiplicity
of infection of 0.1, and a 10-fold excess of uninfected
Bax+/+ cells was added after 24 h of
infection. After an additional 2 days of infection, the yield of
chlamydiae from adherent cells and cells in suspension was measured by
titrating on uninfected HeLa cells (see "Experimental Procedures").
IFU, inclusion-forming units.
/
cells were infected with C. muridarum for 2 days, and apoptosis was measured. No difference
was observed in sensitivity to apoptosis of wild-type and
Bid-deficient cells (Fig. 1A), suggesting that BAX activation is initiated within the interior of the infected cell.
/
cells were dying through necrosis more often than
Bax+/+ cells after a 2-day infection. Whereas
30% of the cells were apoptotic and 10% were necrotic in the
Bax+/+ population, 7% were apoptotic and 34%
were necrotic in the Bax
/
population (Fig.
1B).
/
cells were infected for 3 days, and the
bacteria were harvested from supernatant and infected cells. The
recovered bacteria were then used to reinfect wild-type cells, and the
efficiency of infection was evaluated by immunofluorescence. A larger
number of infectious chlamydiae were recovered from the
Bax+/+ than the Bax
/
cells (Fig. 1C), suggesting that the bacteria may use
apoptosis to exit from cells at the end of the first infection cycle
before beginning a new round of infection. To rule out the possibility that Bax deficiency may be inhibiting growth of
intracellular chlamydiae, the number of infectious vacuoles was also
measured after a 24-h infection, before any apoptosis is observed; the infection at 24 h was the same in either
Bax+/+ or Bax
/
cells
(not shown). Since fibroblasts and epithelial cells express a PS
receptor (56) that could be used to phagocytose
Chlamydia-containing apoptotic bodies, these results suggest
that Chlamydia may use apoptosis to release infectious
bacteria from infected host cells in order to initiate a new infection cycle.
/
mice. The mouse model of C. muridarum infection of the
female genital tract mimics human infection (2, 9, 10) and is a useful
model for Chlamydia infection and adaptive immunity to infection. Bax-deficient mice are also convenient for
studies on Chlamydia infection, since the mice are healthy,
the levels of the antiapoptotic molecules BCL2 and BCL-XL
are unaffected, and the distribution of different lymphocyte
populations (CD4
CD8
,
CD4+CD8+, CD4+, and
CD8+ cells) are unaltered, compared with
Bax+/+ mice (57). The infection was less
efficient and disappeared more quickly in the
Bax
/
mice than in control
Bax+/+ mice (Fig.
2), consistent with a role for
BAX-dependent apoptosis in the propagation of chlamydiae
in vivo.
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Fig. 2.
Chlamydial infection is decreased in
Bax /
mice. A, intensity and duration of primary lower
genital tract infection. Female Bax+/+
(open circles) and
Bax
/
(closed circles)
mice were infected with C. muridarum, and the course of
infection was monitored by cervico-vaginal swabbing. p < 0.001 by two-way analysis of variance for
Bax+/+ versus
Bax
/
. B, elimination of
chlamydiae from wild-type and Bax
/
mice.
Results are expressed as the percentage of animals positive for
infection over time. Bax
/
mice
(closed circles) resolved the infection more
rapidly than Bax+/+ mice (open
circles) with all of the Bax
/
mice being negative for infection by day 16. In contrast, 4 of 10 Bax+/+ mice were still positive for infection on
day 20. IFU, inclusion-forming units.
, and of the murine CXC
chemokine, macrophage inflammatory protein 2 (14, 50). These responses routinely peak during the first week of infection and decline toward
base line during the second week. Enzyme-linked immunosorbent assay
measurement of cytokines in genital tract secretions revealed similar
kinetics in the Bax+/+ and
Bax
/
mice in this study (Fig.
3). However, the proinflammatory
mediators were significantly increased during the first week of
infection in the Bax
/
mice compared with the
Bax+/+ mice. Further, we detected extremely high
levels of IFN-
, a protein with marked antichlamydial effects, in the
Bax
/
mice compared with
Bax+/+ mice during the first week of infection
(Fig. 3). The detection of higher levels of inflammatory mediators in
the Bax
/
mice compared with the
Bax+/+ mice is made more significant by the fact
that the infection was much less efficient in the
Bax
/
mice. These data are consistent with
increased cell death by necrosis during chlamydial infection in the
absence of BAX.
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Fig. 3.
Secretion of inflammatory proteins is
increased in
Bax /
mice. TNF-
(A) macrophage-inflammatory protein 2 (MIP-2) (B), and IFN-
(C) levels
were significantly increased in the Bax
/
(closed circles) mice compared with
Bax+/+ (open circles)
during the first week of infection. Genital tract secretions were
eluted from vaginal sponges collected from individual animals before
and after infection. Results are expressed as mean and S.E. of cytokine
measurements from five animals.
/
mice. Moderate to severe inflammation
was detected in the endocervix and uterine horns with a predominance of
polymorphonuclear neutrophils (PMNs) but high numbers of lymphocytes
also being seen. By immunohistochemical staining, the median
inflammatory score for PMNs was 4 for Bax
/
and 3 for Bax+/+ on day 7 (p = 0.375), and for lymphocytes it was 2.0 for
Bax
/
and for Bax+/+
(p = 0.5) (analysis of variance on ranks). Most of the
lymphocytes were CD4+ in both groups, with comparatively
low numbers of CD8+ cells being found (median score for
CD4+ T cells = 2.0 for both groups on day 7;
CD8+ T cells = 1). Mild to moderate inflammation was
detected in the oviducts in both Bax+/+ and
Bax
/
mice, again with a predominance of PMNs
being found. Tissues from mice sacrificed on day 24, at a time when
infection had mostly resolved, revealed equal numbers of acute (PMNs)
and chronic inflammatory cells (lymphocytes) in
Bax+/+ and Bax
/
mice.
However, in 4 of 5 Bax
/
mice, large
granulomatous nodules with marked central necrosis were found
scattered throughout 9 of 10 uterine horns (Fig.
4). These nodules were seen in only 1 of
10 horns from Bax+/+ mice; p = 0.001, Fisher exact test). Thus, although infection is less efficient
in Bax
/
mice, it results in greater
release of inflammatory mediators and increased chronic tissue
pathology.
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Fig. 4.
Large granulomas are prevalent in C. muridarum-infected
Bax /
mice. A, histopathological examination of hematoxylin-
and eosin-stained longitudinal sections from the uterine horns
of mock-infected Bax
/
mice revealed
normal endometrial type glands and an absence of inflammation. Uteri
from mock-infected Bax+/+ mice were also normal
(not shown). B, whereas the uteri from C. muridarum-infected Bax+/+ mice revealed a
paucity of granulomas, the horns were dilated with PMNs within the
lumen as well as the glandular epithelium and scattered lymphocytes and
plasma cells in the stroma. C, the uterine horns from
C. muridarum-infected Bax
/
mice
revealed multiple areas of granulomatous inflammation with aggregates
of large cells containing abundant eosinophilic cytoplasm consistent
with histiocytes and scattered small lymphocytes.
/
and Bax+/+
mice. Both groups demonstrated high titers of IgG2a and low titers of
IgG1 (Fig. 5), demonstrating that a
TH1 response was stimulated in both cases. Both
Bax+/+ and Bax
/
were
also completely resistant to reinfection when challenged 70 days after
primary vaginal inoculation (not shown). Thus, despite the increased
release of inflammatory mediators and enhanced pathology after primary
infection in Bax
/
mice, the absence of Bax
did not affect the quality or magnitude of the acquired immune
response.
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Fig. 5.
The antibody response in
Bax+/+ and
Bax /
mice. The acquired immune response, as determined by antibody
titers in serum, was similar in both groups of mice. Black
bar, IgG1 in Bax+/+; gray
bar, IgG2a in Bax+/+; dark
gray bar with diagonal
lines, IgG1 in Bax
/
;
light gray bar with
diagonal lines, IgG2a in
Bax
/
. Data are expressed as the mean titer
(log10) + S.E. for five mice at each time point. **,
significantly higher titers of IgG2a compared with IgG1 for
Bax+/+ and Bax
/
,
p > 0.005. Bax+/+ and
Bax
/
titers were not significantly different
at any time point.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice than in
Bax+/+ mice also reinforces the interpretation
that BAX-dependent apoptosis could facilitate chlamydial
propagation. Finally, Bid
/
cells are as
sensitive to Chlamydia-induced apoptosis as
Bid+/+ cells. Ligation of the Fas or TNFR1 death
receptors on the cell surface leads to cleavage of BID, which activates
BAX (47). The lack of involvement of BID during Chlamydia
infection suggests that BAX activation is initiated within the host
cell. Activation could be related to infection-related metabolic stress
(58), or it could be triggered by signals released from the chlamydial vacuole via type 3 secretion mechanisms (59-61). Activation of BAX is
clearly advantageous for Chlamydia, and it is tempting to
speculate that other intracellular microbes may use BAX-mediated apoptosis to enhance their propagation. These results thus reveal a
novel function for a host cell proapoptotic protein, which until now
has been known to promote apoptosis through induction of mictochondrial dysfunction and whose singular deficiency in mice results in only minor
changes to the immune system (57).
/
mice, the secretion of inflammatory
cytokines was higher in Bax
/
than in
wild-type mice. The secretion of TNF-
, IFN-
, and the murine
equivalent of IL-8, macrophage inflammatory protein 2, have been
previously reported during C. muridarum infection, but until
now the extent of their secretion has always correlated with the
intensity of infection (8, 13, 64). Whereas several interpretations of these data could be envisioned, we propose that
apoptosis of infected cells in Bax
/
mice is
postponed, causing the cells to die of necrosis more often than in
Bax+/+ mice. This explanation is consistent with
the observation that more necrotic cells are observed when
Bax-deficient cells are infected in vitro than
when wild-type cells are infected. Phagocytosis of apoptotic cells by
macrophages leads to secretion of anti-inflammatory cytokines such as
IL-10 and transforming growth factor-
, but necrotic cells
stimulate secretion of proinflammatory cytokines, including TNF-
,
IL-1
, and IL-8 (65-67). Although these possibilities are not
mutually exclusive, the resulting increase in IFN-
observed in
Bax
/
mice may also contribute to their
faster resolution of infection. IFN-
is a known inducer of aberrant
forms of Chlamydia in vitro; the cytokine
adversely affects normal growth and division of reticulate bodies and
interrupts their redifferentiation into infectious EB (68). IFN-
induction of aberrant, noninfectious forms of Chlamydia may
thus contribute to reduced infection in the
Bax
/
mice.
/
mice
reinforces the notion that secretion of inflammatory cytokines by
infected epithelial cells and neighboring macrophages may be responsible for the chronic tissue damage associated with
Chlamydia infection. Although the hallmark of both ocular
(trachoma) and urogenital chlamydial infections is the development of
lymphoid follicles (69-72), granulomas have occasionally been reported
in human (73), non-human primate (74), murine (75), and veterinary disease (76). Loss of function mutations in Bax have been
reported in humans and may be associated with increased incidence and
progression of cancer (77-80). Our data suggest that mutations in
Bax might lead to an increase in the severity of chlamydial
genital tract disease. This is the first report of the effect of
Bax mutation in an infectious disease model.
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ACKNOWLEDGEMENTS |
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We are grateful to Thomas Jungas and Jim Sikes for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by the Institut Pasteur (PTR 60), INSERM, Université Paris 7, National Institutes of Health Grant AI054624, and the Bates-Wheeler Foundations, Arkansas Children's Hospital Research Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a fellowship from the Fondation pour la Recherche Médicale.
¶ These two authors share senior authorship.
To whom correspondence should be addressed: Institut Jacques
Monod, Universite Paris 7, 2 place Jussieu, Tour 43, 75251 Paris cedex
05, France. Fax: 33-1-44275265; E-mail: ojcius@noos.fr.
Published, JBC Papers in Press, December 31, 2002, DOI 10.1074/jbc.M211275200
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ABBREVIATIONS |
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The abbreviations used are: MoPn, C. trachomatis mouse pneumonitis strain; EB, elementary body; IL, interleukin; TNF, tumor necrosis factor; PS, phosphatidylserine; PI, propidium iodide; PMN, polymorphonuclear neutrophils; IFN, interferon.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Schachter, J., and Caldwell, H. D. (1980) Annu. Rev. Microbiol. 34, 285-310[CrossRef][Medline] [Order article via Infotrieve] |
2. | Bavoil, P. M., Hsia, R.-c., and Rank, R. G. (1996) Bull. Inst. Pasteur 94, 5-54[CrossRef] |
3. | Campbell, L. A., Kuo, C. C., and Grayston, J. T. (1998) Emerg. Infect. Dis. 4, 571-579[Medline] [Order article via Infotrieve] |
4. | Gerbase, A. C., Rowley, J. T., and Mertens, T. E. (1998) Lancet 351, 2-4[CrossRef][Medline] [Order article via Infotrieve] |
5. | Washington, A. E., Johnson, R. E., and Sanders, L. L., Jr. (1987) JAMA (J. Am. Med. Assoc.) 257, 2070-2072[Abstract] |
6. | Everett, K. D. E., Bush, R. M., and Andersen, A. A. (1999) Int. J. Syst. Bacteriol. 49, 415-440[Abstract] |
7. |
Schachter, J.,
Stephens, R. S.,
Timms, P.,
Kuo, C.,
Bavoil, P. M.,
Birkelund, S.,
Boman, J.,
Caldwell, H.,
Campbell, L. A.,
Chernesky, M.,
Christiansen, G.,
Clarke, I. N.,
Gaydos, C.,
Grayston, J. T.,
Hackstadt, T.,
Hsia, R.,
Kaltenboeck, B.,
Leinonnen, M.,
Ojcius, D.,
McClarty, G.,
Orfila, J.,
Peeling, R.,
Puolakkainen, M.,
Quinn, T. C.,
Rank, R. G.,
Raulston, J.,
Ridgeway, G. L.,
Saikku, P.,
Stamm, W. E.,
Taylor-Robinson, D.,
Wang, S.-P.,
and Wyrick, P. B.
(2001)
Int. J. Syst. Evol. Microbiol.
51,
249 |
8. | Rank, R. G. (1999) in Chlamydia: Intracellular Biology, Pathogenesis, and Immunity (Stephens, R. S., ed) , pp. 239-295, American Society for Microbiology Press, Washington, D. C. |
9. | Barron, A. L., White, H. J., Rank, R. G., Soloff, B. L., and Moses, E. B. (1981) J. Infect. Dis. 143, 63-66[Medline] [Order article via Infotrieve] |
10. | de la Maza, L. M., Pal, S., Khamesipour, A., and Peterson, E. M. (1994) Infect. Immun. 62, 2094-2097[Abstract] |
11. | Rank, R. G., and Sanders, M. M. (1992) Am. J. Pathol. 140, 927-936[Abstract] |
12. | Toth, M., Jeremias, J., Ledger, W. J., and Witkin, S. S. (1992) Surg. Gynecol. Obstet. 174, 359-362[Medline] [Order article via Infotrieve] |
13. | Darville, T., Laffoon, K. K., Kishen, L. R., and Rank, R. G. (1995) Infect. Immun. 63, 4675-4681[Abstract] |
14. | Darville, T., Andrews, C. W., Laffoon, K. K., Shymasani, W., Kishen, L. R., and Rank, R. G. (1997) Infect. Immun. 65, 3064-3073 |
15. | Williams, D. M., Bonewald, L. F., Roodman, G. D., Byrne, G. I., Magee, D. M., and Schachter, J. (1989) Infect. Immun. 57, 1351-1355[Medline] [Order article via Infotrieve] |
16. | Rockey, D. D., and Matsumoto, A. (1999) in Prokaryotic Development (Brun, Y. V. , and Shimkets, L. J., eds) , pp. 403-425, American Society for Microbiology Press, Washington, D. C. |
17. | Moulder, J. W. (1991) Microbiol. Rev. 55, 143-190[Medline] [Order article via Infotrieve] |
18. | Gao, L.-Y., and Abu Kwaik, Y. (2000) Trends Microbiol. 8, 306-313[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Galvan, V.,
and Roizman, B.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
3931-3936 |
20. | Chen, X. M., Gores, G. J., Paya, C. V., and LaRusso, N. F. (1999) Am. J. Physiol. 277, G599-G608[Medline] [Order article via Infotrieve] |
21. | Chen, X. M., Levine, S. A., Splinter, P. L., Tietz, P. S., Ganong, A. L., Jobin, C., Gores, G. J., Paya, C. V., and LaRusso, N. F. (2001) Gastroenterology 120, 1774-1783[Medline] [Order article via Infotrieve] |
22. |
Fan, T.,
Lu, H.,
Shi, L.,
McCarthy, G. A.,
Nance, D. M.,
Greenberg, A. H.,
and Zhong, G.
(1998)
J. Exp. Med.
187,
487-496 |
23. |
Fischer, S. F.,
Schwarz, C.,
Vier, J.,
and Hacker, G.
(2001)
Infect. Immun.
69,
7121-7129 |
24. |
Dean, D.,
and Powers, V. C.
(2001)
Infect. Immun.
69,
2442-2447 |
25. |
Rajalingam, K.,
Al-Younes, H.,
Muller, A.,
Meyer, T. F.,
Szczepek, A. J.,
and Rudel, T.
(2001)
Infect. Immun.
69,
7880-7888 |
26. | Gibellini, D., Panaya, R., and Rumpianesi, F. (1998) Zentralblatt für Bakteriologie 288, 35-43[Medline] [Order article via Infotrieve] |
27. |
Perfettini, J.-L.,
Darville, T.,
Gachelin, G.,
Souque, P.,
Huerre, M.,
Dautry-Varsat, A.,
and Ojcius, D. M.
(2000)
Infect. Immun.
68,
2237-2244 |
28. |
Perfettini, J. L.,
Reed, J. C.,
Israël, N.,
Martinou, J. C.,
Dautry-Varsat, A.,
and Ojcius, D. M.
(2002)
Infect. Immun.
70,
55-61 |
29. |
Ojcius, D. M.,
Souque, P.,
Perfettini, J. L.,
and Dautry-Varsat, A.
(1998)
J. Immunol.
161,
4220-4226 |
30. |
Perry, L. L.,
Feilzer, K.,
Hughes, S.,
and Caldwell, H. D.
(1999)
Infect. Immun.
67,
1379-1385 |
31. | Nagata, S. (1997) Cell 88, 355-365[Medline] [Order article via Infotrieve] |
32. | Kroemer, G., Dallaporta, B., and Resche-Rigon, M. (1998) Annu. Rev. Physiol. 60, 619-642[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Adams, J. M.,
and Cory, S.
(1998)
Science
281,
1322-1326 |
34. | Shimizu, S., Narita, M., and Tsujimoto, Y. (1999) Nature 399, 483-487[CrossRef][Medline] [Order article via Infotrieve] |
35. | Rossé, T., Olivier, R., Monney, L., Rager, M., Conus, S., Fellay, I., Jansen, B., and Borner, C. (1998) Nature 391, 441-442[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Deas, O.,
Dumont, C.,
MacFarlane, M.,
Rouleau, M.,
Hebib, C.,
Harper, F.,
Hirsch, F.,
Charpentier, B.,
Cohen, G. M.,
and Senik, A.
(1998)
J. Immunol.
161,
3375-3383 |
37. |
Xiang, J.,
Chao, D. T.,
and Korsmeyer, S. J.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
14559-14563 |
38. |
McCarthy, N. J.,
Whyte, M. K. B.,
Gilbert, C. S.,
and Evan, G. I.
(1997)
J. Cell Biol.
136,
215-227 |
39. |
Pastorino, J. G.,
Chen, S. T.,
Tafani, M.,
Snyder, J. W.,
and Farber, J. L.
(1998)
J. Biol. Chem.
273,
7770-7775 |
40. | Salvesen, G. S., and Dixit, V. M. (1997) Cell 91, 443-446[Medline] [Order article via Infotrieve] |
41. |
Hersh, D.,
Monack, D. M.,
Smith, M. R.,
Ghori, N.,
Falkow, S.,
and Zychlinsky, A.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
2396-2401 |
42. |
Hilbi, H.,
Moss, J. E.,
Hersh, D.,
Chen, Y.,
Arondel, J.,
Banerjee, S.,
Flavell, R. A.,
Yuan, J.,
Sansonetti, P. J.,
and Zychlinsky, A.
(1998)
J. Biol. Chem.
273,
32895-32900 |
43. |
Lu, H.,
Shen, C.,
and Brunham, R. C.
(2000)
J. Immunol.
165,
1463-1469 |
44. | La Verda, D., and Byrne, G. I. (1994) Immunol. Ser. 60, 381-399[Medline] [Order article via Infotrieve] |
45. | Hogquist, K. A., Nett, M. A., Unanue, E. R., and Chaplin, D. D. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 8485-8489[Abstract] |
46. | Golstein, P., Ojcius, D. M., and Young, J. D. (1991) Immunol. Rev. 121, 29-65[Medline] [Order article via Infotrieve] |
47. | Wei, M. C., Zong, W.-X., Cheng, E. H.-Y., Lindsten, T., Panoutsakopoulou, V., Ross, A. J., Roth, K. A., MacGregor, G. R., Thompson, C. B., and Korsmeyer, S. J. (2001) Science 2, 727-730[CrossRef] |
48. | Perfettini, J.-L., Gissot, M., Souque, P., and Ojcius, D. M. (2002) Methods Enzymol. 358, 334-344[Medline] [Order article via Infotrieve] |
49. | Nicoletti, I., Migliorati, G., Pagliacci, M. C., Grignani, F., and Riccardi, C. (1991) J. Immunol. Methods 139, 271-279[CrossRef][Medline] [Order article via Infotrieve] |
50. |
Darville, T.,
Andrews, C. W.,
Sikes, J. D.,
Fraley, P. L.,
and Rank, R. G.
(2001)
Infect. Immun.
69,
3556-3561 |
51. | Ramsey, K. H., Newhall, W. J., and Rank, R. G. (1989) Infect. Immun. 57, 2441-2446[Medline] [Order article via Infotrieve] |
52. |
Morrison, S. G.,
and Morrison, R. P.
(2000)
Infect. Immun.
68,
2870-2879 |
53. |
Vercammen, D.,
Brouckaert, G.,
Denecker, G.,
Van de Craen, M.,
Declercq, W.,
Fiers, W.,
and Vandenabeele, P.
(1998)
J. Exp. Med.
188,
919-930 |
54. | Holler, N., Zaru, R., Micheau, O., Thome, M., Attinger, A., Valitutti, S., Bodmer, J.-L., Schneider, P., Seed, B., and Tschopp, J. (2000) Nat. Immunol. 1, 489-495[CrossRef][Medline] [Order article via Infotrieve] |
55. |
Matsumura, H.,
Shimizu, Y.,
Ohsawa, Y.,
Kawahara, A.,
Uchiyama, Y.,
and Nagata, S.
(2000)
J. Cell Biol.
151,
1247-1256 |
56. | Fadok, V., Bratton, D. L., Rose, D. M., Pearson, A., Ezekewitz, R. A. B., and Henson, P. M. (2000) Nature 405, 85-90[CrossRef][Medline] [Order article via Infotrieve] |
57. | Knudson, C. M., Tung, K. S. K., Tourtellotte, W. G., Brown, G. A. J., and Korsmeyer, S. J. (1995) Science 270, 96-99[Abstract] |
58. |
Bavoil, P. M.,
Hsia, R.,
and Ojcius, D. M.
(2000)
Microbiology
146,
2723-2731 |
59. | Hsia, R., Pannekoek, Y., Ingerowski, E., and Bavoil, P. M. (1997) Mol. Microbiol. 5, 351-359 |
60. | Stephens, R. S., Kalman, S., Lammel, C., Fan, J., Marathe, R., Aravind, L., Mitchell, W., Olinger, L., Tatusov, R. L., Zhao, Q., Koonin, E. V., and Davis, R. W. (1998) Science 23, 638-639[CrossRef] |
61. | Fields, K. A., and Hackstadt, T. (2000) Mol. Microbiol. 38, 1048-1060[CrossRef][Medline] [Order article via Infotrieve] |
62. | Savill, J., and Fadok, V. (2000) Nature 407, 784-788[CrossRef][Medline] [Order article via Infotrieve] |
63. | Platt, N., da Silva, R. P., and Gordon, S. (1998) Trends Cell Biol. 8, 365-372[CrossRef][Medline] [Order article via Infotrieve] |
64. |
Rasmussen, S. J.,
Eckmann, L.,
Quayle, A. J.,
Shen, L.,
Zhang, Y. X.,
Anderson, D. J.,
Fierer, J.,
Stephens, R. S.,
and Kagnoff, M. F.
(1997)
J. Clin. Invest.
99,
77-87 |
65. | Gallucci, S., Lolkema, M., and Matzinger, P. (1999) Nat. Med. 5, 1249-1255[CrossRef][Medline] [Order article via Infotrieve] |
66. |
Fadok, V. A.,
Bratton, D. L.,
Konowal, A.,
Freed, P. W.,
Westcott, J. Y.,
and Henson, P. M.
(1998)
J. Clin. Invest.
101,
890-898 |
67. | Voll, R. E., Herrmann, M., Roth, E. A., Stach, C., Kalden, J. R., and Girkontaite, I. (1997) Nature 390, 350-351[CrossRef][Medline] [Order article via Infotrieve] |
68. | Shemer, Y., and Sarov, I. (1985) Infect. Immun. 48, 592-596[Medline] [Order article via Infotrieve] |
69. | Kiviat, N. B., Wolner-Hanssen, P., Eschenbach, D. A., Wasserheit, J. N., Paavonen, J. A., Bell, T. A., Critchlow, C. W., Stamm, W. E., Moore, D. E., and Holmes, K. K. (1990) Am. J. Surg. Pathol. 14, 167-175[Medline] [Order article via Infotrieve] |
70. | el-Asrar, A. M., Van den Oord, J. J., Geboes, K., Missotten, L., Emarah, M. H., and Desmet, V. (1989) Br. J. Ophthalmol. 73, 276-282[Abstract] |
71. | Hare, M. J., Toone, E., Taylor-Robinson, D., Evans, R. T., Furr, P. M., Cooper, P., and Oates, J. K. (1981) Br. J. Obstet. Gynaecol. 88, 174-180[Medline] [Order article via Infotrieve] |
72. | Paavonen, J., Vesterinen, E., Meyer, B., and Saksela, E. (1982) Obstet. Gynecol. 59, 712-715[Abstract] |
73. | Christie, A. J., and Krieger, H. A. (1980) Am. J. Obstet. Gynecol. 136, 958-960[Medline] [Order article via Infotrieve] |
74. | Quinn, T. C., Taylor, H. R., and Schachter, J. (1986) J. Infect. Dis. 154, 833-841[Medline] [Order article via Infotrieve] |
75. |
Yang, X.,
Gartner, J.,
Zhu, L.,
Wang, S.,
and Brunham, R. C.
(1999)
J. Immunol.
162,
1010-1017 |
76. | Jones, G. E., Jones, K. A., Machell, J., Brebner, J., Anderson, I. E., and How, S. (1995) Vaccine 13, 715-723[CrossRef][Medline] [Order article via Infotrieve] |
77. | Bandoh, N., Hayashi, T., Kishibe, K., Takahara, M., Imada, M., Nonaka, S., and Harabuchi, Y. (2002) Cancer 94, 1968-1980[CrossRef][Medline] [Order article via Infotrieve] |
78. | LeBlanc, H., Lawrence, D., Varfolomeev, E., Totpal, K., Morlan, J., Schow, P., Fong, S., Schwall, R., Sinicropi, D., and Ashkenazi, A. (2002) Nat. Med. 8, 274-281[CrossRef][Medline] [Order article via Infotrieve] |
79. | Mullauer, L., Gruber, P., Sebinger, D., Buch, J., Wohlfart, S., and Chott, A. (2001) Mutat. Res. 488, 211-231[CrossRef][Medline] [Order article via Infotrieve] |
80. |
Ionov, Y.,
Yamamoto, H.,
Krajewski, S.,
Reed, J. C.,
and Perucho, M.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
10872-10877 |
81. |
Gershov, D.,
Kim, S.,
Brot, N.,
and Elkon, K. B.
(2000)
J. Exp. Med.
192,
1353-1364 |
82. | Rosen, A., and Casciola-Rosen, L. (1999) Cell Death Differ. 6, 6-12[CrossRef][Medline] [Order article via Infotrieve] |